methods cell biology 2015
DESCRIPTION
Presentation of Johannes A. Schmidwww.meduniwien.ac.at/user/johannes.schmidsome contents contributed by:Dr. Lukas MachInstitut für Angewandte Genetik und Zellbiologie ,Univ. f. BodenkulturTRANSCRIPT
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Methods in Cell Biology 2015Johannes A. Schmid
Internet: www.meduniwien.ac.atuser!ohannes.schmid
some contents contri"uted "y:
#r. $u%as Mach
Institut &'r Angewandte (eneti% und )ell"iologie *+ni,. &. Boden%ultur
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-,er,iew o& o/ics
scienti&ic strategies and assay systems
cell culture
la"elling and trans&ection o& cells
analysis o& cellular com/onents
analysis o& molecular interactions
&luorescence measurements
microsco/y
&low analysis &luorescence acti,ated cell sorting* ACS3
analysis o& ,arious cellular /rocesses /roli&eration* a/o/tosis..3
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#etails o& the lecture
1. Scienti&ic strategies2. cell culture4. la"elling and trans&ection o& cells
a3 radioacti,e and chemical la"elling"3 trans&ections: o,ere/ression o& genes and gene su//ressionc3 re/orter gene assays
6. gene7su//ression si89A7technologies35. analysis o& #9A and /roteins electro/horesis and "lotting3. su"cellular &ractionation centri&ugations;3<. methods to detect macromolecular interactions
a3 =east 17 und 27hy"rid systems"3 co7immuno/reci/itationsc3 &luorescence resonance energy trans&er 8>3
?. methods o& &luorescence measurements@. realtime7C8 10. transmitted light microsco/y and contrast /rinci/les11. &luorescence microsco/y12. con&ocal laser scanning microsco/y14. &low analysis ACS316. analysis o& ,arious cellular /rocesses /roli&eration* a/o/tosis..315. methods to in,estigate ,esicular trans/ort /rocesses
9odes o& regulation in cellular systems
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#9A
/re7m89A m89A
degradation
degradation
/osttranslationalmodi&ication
translation
transcri/tion
trans/ort
s/licing
transcri/tion
&actor
trans/ortm89A
/re7micro789A micro789A
/rocessingmicro789A
acti,ation
signal transduction
/oly7u"iuitination
nucleus
>8
cell mem"ranerece/torsligands
(olgi
trans/ort
/rotein
trans/ort
signaltransduction
oligomeriation
modi&ied/rotein
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(eneral scienti&ic strategies
descri/ti,e strategies: the whole system cell* organism3 iso"ser,ed without in&luencing it
ad,antage: /hysiological states are not altereddisad,antage: it is di&&icult to elucidate cause7e&&ect relationshi/s
mechanistic mani/ulating3 strategies: ,arious &actors are %e/tconstant* while others are altered on /ur/ose D the change inthe whole system is monitored.
ad,antage : cause7e&&ect relationshi/s can "e monitored or
detecteddisad,antage : the /hysiological steady state is altered andin&luenced. 8esults might "e arte&acts o& the measurementsystem.
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>/erimental Systems I
In ,itro* "iochemical systems: in,estigation in a solutione.g. enyme reactions3: many /arameters can "e &iedtem/erature* /E* "u&&er com/osition;3.>am/le: enyme acti,ity assay* >MSA ;
In ,itro* cell "iological systems: including cellular structures
e.g. in ,itro7transcri/tiontranslation with mem"ranecom/onents* in ,itro7&usion o& endosomes* nuclear im/ortassays with isolated nuclei etc. >/erimental conditionsha,e to "e set in a way that cellular structures are notdamaged e.g. isotonic "u&&er* /hysiological /E;.3F reaction/artners can "e in&luenced widely e.g. anti"odies can "eadded;3
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>/erimental Systems II
cell culture systems7 immortalied cell lines: unlimited /assages
7 /rimary cells: limited /assage num"er* morecom/licated cell culture e.g. coated /lates;3
7 co7culture o& di&&erent cell ty/es:e.g. %eratinocytes G &i"ro"lasts in a collagen matri.
xenograft systems:- cells are in!ected into immuno7com/romied micenude mice* SCI# mice3F e.g. su"cutaneously7 tissue recom"ination systems e.g. /rostate e/ithelialcells with mesenchymal cells in!ected into the renalca/sule o& SCI# mice3
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Organ culturese.g. s%in sheets*"rain slices;3
Organ perfusions
>/erimental Systems III
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Animal Experiments:
Just the whole organism /ro,ides the &ull com/le "iological system that is
rele,ant &or most "iomedical research to/ics. he whole organism includes
su/erordinated systems such as the ner,ous system* the "lood circulation*
the endocrine system and so on. he cells are in their normal organ
en,ironmentF cellular communications are intact;. D thus the highest
/ossi"le /hysiological state can "e achie,ed. Eowe,er* s/eci&ic com/onents
o& the system e.g. certain cells3 are not easily accessi"le D and s/eci&ic
e/erimental mani/ulations e.g. o& s/eci&ic cells without side e&&ects3 are
o&ten di&&icult3. Cause7e&&ect relationshi/s are o&ten di&&icult to elucidate D
and there is a "ig H"lac% "o due to the com/leity o& the system.
It has to "e considered that results o"tained with animals such as mice o&ten
cannot "e trans&erred to human "eings des/ite the highly /hysiological
system.
>/erimental Systems I
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>/erimental Systems
Special case: Transgene and knock-out animals :S/eci&ic elimination o& genes %noc%7out3 or incor/oration o& genestransgenes* %noc%7in3 allow a "etter elucidation o& cause7e&&ect relationshi/s.Eowe,er* classical %noc%7outs eliminating a gene in all cells3 D is o&tenem"ryonically lethal D and does not allow conclusions &or its &unction in theadult animal e.g. mouse3. ice ,ersa* it can ha//en that there is no/henoty/e i& the &unction o& the gene is ta%en o,er "y another gene3. Knoc%7ins can "e uite arti&icial as well i& the transgene is e/ressed at higher le,els
with strong /romoters3. Modern a//roaches o&ten aim &or Hconditional %noc%7outs or %noc%7ins: In most cases the Cre7recom"inase lo system is used&or that /ur/ose:Conditional %noc%7out: the gene or a crucial eon3 is /laced "etween losites D Cre recom"inase which can "e e/ressed in s/eci&ic organs or cells "yorgan s/eci&ic /romoters3 cuts out the gene D thus the gene is deleted !ust ina certain organ or cell ty/eF using induci"le Cre* this system allows genedeletion at a de&ined time /oint e.g. when the animal is adult3.Conditional %noc%7in: the gene is /laced "ehind a Sto/7cassette* which is&lan%ed "y lo sites. Lithout Cre acti,ity* the gene is not e/ressed* with Creacti,ity organ s/eci&ic3* the Sto/ cassette is ecised and the gene is e/ressed
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Conditional transgene mouse models with the
Cre lo system
Cre recom"inase cuts outseuences "etween lo sitesor in,erts seuences "etweenin,erted lo sites3
Cre e/ression can "erendered cell7ty/e or organ7s/eci&ic using cell7ty/e s/eci&ic/romoters dri,ing Cree/ression s/atial control3
Cre e/ression can "e madeinduci"le "y using chimeras o&Cre with mutated estrogenrece/tor domainstem/oral control:e.g. Cre7>823
>ndogenous genes can "e&lan%ed "y lo sites usingrecom"ination techniues De.g. a&&ecting essential eons3 conditional %noc%7out
(enes can "e o,ere/ressedconditionally "y inserting ane/ression construct headed
"y a lo7&lan%ed HSto/cassette* which is cut out "yCre recom"inase
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1. Cre mouse strain2. lox-mouse strain
>am/les &or conditional mouse models
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Crem>8 m>8
mE! : mutated estrogen rece/tor res/onds to tamoi&en as ligandF withoutthe ligand it %ee/s the Cre in the cytosol inacti,eF u/on addition o& tamoi&en* the nuclear localiation seuences o& m>8 "ecome acti,e leading
to translocation o& the chimera into the nucleus* where Cre recom"inase caneert its &unction on lo7&lan%ed #9A seuences3.
cell7ty/e s/eci&ic/romoter
Cre7>8 2
Sto/ (ene o& interest(ene o& interest
lo lo good /romotere.g. /CA((S3
Crem>8 m>8
amam
Crem>8 m>8
amam
conditional knock-out conditional transgene
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S/eci&ic cell a"lation or cell la"eling
in transgenic mice
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Sto/ #8 #i/hteria toin rece/tor3
good /romotere.g. /CA((S3
lo lo
cross7"reeding with a cell7ty/e s/eci&ic Crestrain
#8 is e/ressed only in s/eci&ic cell ty/es
in!ection o& di/hteria toin leads to s/eci&ic
%illing o& these cells
Sto/ >= enhanced yellow &luorescent /rotein3
good /romotere.g. /CA((S3
lo lo
&luorescent la"eling o& a s/eci&ic cell ty/e o& interest
(enome >diting &or generating transgeneanimals
9o,el methods to edit genes directly in the genome* e.g. usingC8IS8Cas@ technology or )n7&inger nucleases* or A$>9sallow &aster and e,en multi/leing ty/e mani/ulations e.g.targeting 5 genes simultaneously3
(enome editing is then usually done in >S7cells* which are
su"seuently in!ected into "lastocysts.
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Cell Culture Methods cell culture o& immortalied cells
cell culture o& /rimary cells o&ten !ust a &ew /assages3
culture o& /olaried cells
co7culture o& di&&erent cell ty/es
/rimary &i"ro"lasts&rom s%in dermis3
trans&ormed&i"ro"lasts
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Lhat you need &or cell culture
Incu"ator 4<NC3 with C-27su//ly and humidi&ication
Sterile "ench $aminar low: alaminar &low o& &iltered air %ee/sthe "ench sterile3 D has to "eswitched on a//ro. 10 min
"e&ore using it* the &ilter has to"e chec%ed &rom time to timearticle Measurement3
in,erted cell culture microsco/e6* 10* 20 o"!ecti,es3
$27"iosa&ety e.g. &or ,irus7wor%3: not only incoming air is&iltered* "ut also the air thatlea,es the laminar &low.
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mammalian cells need media with ,itamins* aminoacids* hormones and growth &actors
Serum* mostly &etal cal& serum CS3* is the source o&growth &actors such as (* >(;3
Common com/osition7 #M>M #ul"eccoOs Modi&ied >ssential Medium37 10P CS7 2 mM (lutamine unsta"le amino acid* has to "e addedagain* i& the medium is older than a//. wee%s3
7 enicillin 100 uml37 Stre/tomycin 100 Qgml3
Culture Media
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has high concentrations o& growth &actorse.g.: >(* e/idermal growth &actorF (*&i"ro"last growth &actorF I(* insulin7li%egrowth &actor3
low amount o& anti"odies → com/ati"lewith cells o& other s/ecies
com/lement system is inacti,ated "y aheat shoc% 40 min.* 5 °C3
disad,antage o& CS: e/ensi,e ≈ 100 R/er $iter3
might contain contaminants such astetracyclin im/ortant i& you use a et7induci"le cell culture system /urchaseguaranteed et7&ree CS3
Alternati,e sources o& serum: normal cal&serum* horse serum;
etal Cal& Serum CS3 S/ecial (rowthactors
9er,e7 (rowth actor9(3:&or neuronal cells
Ee/atocyte7(rowth
actor E(3: induces celldi,ision o& he/atocytes
Keratinocyte7(rowthactor K(3: &or cultureo& s%in e/ithelial cells%eratinocytes3
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adherent cells ,ersus sus/ension cells
cell culture dishes: usually made hydro/hiliccharged grou/s3 D su&&icient &or most cell ty/es
collagenF (elatine denatured collagen3sometimes necessary &or good attachment e.g. &or/rimary endothelial cells3
Com/onents o& etracellular matri:
i"ronectin* $aminin D o&ten "etter than collagen.
s/ecial case: Heeder7cells irradiated3 D or co7culture o& cells e.g. &i"ro"lasts in a collagen matriwith %eratinocytes on to/3
Adhesion &actors
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Bacteria: might "e a /ro"lem* whenthey are resistant against the anti"ioticsthat are used mostly enStre/3 then you ha,e to use other anti"ioticse.g. %anamycin3
Myco/lasm: /rocaryotes o& an anciente,olutionary stage* which do not ha,e anormal "acterial cell wall there&orethey are resistant against enicillin3can "e eliminated with Kanamycin3
=east: rare might occur when yeastand mammalian cell culture are notstrictly se/arated3
ungi: uite rare
Contaminations in cell culture
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#9A7staining with #AI or Eoechst 4425?
Alternati,e:C87 detection o& Myco/lasm7#9Acommercial %its are a,aila"le3
no Myco/lasm
Myco/lasm
ests &or Myco/lasm7Contamination
+nnoticed myco/lasm contaminations can screw u/ e/erimental results
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Cell culture o& /olaried cells
a/ical side
lateral side
"asal mem"rane
the cells ha,e to "uild thetight !unctions &or "uilding u/the /olarity.
-&ten they are culti,ated on
&ilters* where the two sidesare accessi"le.
tight !unction
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Methods to in,estigate /olaried cells
Model systems
1. sim/le cell culture: !ust one side o& the cell is accessi"le.
2. cell culture on micro/orous mem"ranes: "oth sides aree/erimentally accessi"le* the esta"lishment o& a tight/olaried monolayer can "e chec%ed "e measuring theelectrical resistance "etween the two sides.
4. -rgan cultures: e.g. $i,ing S%in >ui,alent
6. -rgan /er&usion: e.g. /er&usion o& isolated rat li,er
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Culture o& /olaried cells on mem"ranes
Measurement o& electricalresistance
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Culti,ation o& /olaried cells on electrode
cham"ers
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Culti,ation o& /olaried cells on electrodecham"ers
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-rganoty/ic culture: >am/le: S%in >ui,alent
Keratinocytes cells o& the u//er layer o& s%in* the e/idermis3 areseeded onto a collagen matri* which contains &i"ro"lasts dermiscells3. As soon as they "uild a monolayer* they are ele,ated to thesur&ace o& the medium with their u//er side e/osed to air3 D thisinduces cell di&&erentiation and the &ormation o& a /seudo7e/idermiswith se,eral cell layers.
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-rgan er&usion
olaried cells such as he/atocytescan "e maintained in their organarchitecture maintaining their/olarity. he organ is /er&usedusing glas ca/illaries lin%ed to thenormal "lood ,essels the su//lythe organ with "lood and
nutrientsoygen3. A "u&&er 4<°C*/ercolated with -23 containing thenutrients can "e /er&used throughthe li,er. Mar%er su"stances can"e a//lied and their trans/ort&rom the "lood side "asolateral3to the "ile side a/ical3 can "edetermined.
Perfusionsdruck (cm H 2O)
V. porta
Kanüle
Peristaltikpumpe
V. cava Kanüle
Thermosensor
LEBER
Gallengangskanüle
FraktionskollektorComputer
Temperatur-
Messung
Schreiber
93% O2
7% CO2
Wasserbad
Gas-Befeuchtung
Perfusionspuffer
/er&usion /ressure cm E2-3
$i,er
"ile canula
. /orta
. ca,a canula
tem/erature
recording
gas humidi&ication
&raction collector
um/
Later Bath
er&usion Bu&&er
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>am/le o& an e/eriment with /olaried cells
ransmigration o& $eu%ocytes through a Monolayer o& /olariedendothelial cells
$eu%ocytes are la"eled with a&luorescent mar%er e.g. CS>
D an ester* which is turned&luorescent !ust a&ter u/ta%einto cells due to esterases3
Cells are seed onto a layer o& endothelial cells e.g. a&ter acti,ating the
endothelial cells with in&lammatory cyto%ines D which leads to thesynthesis o& adhesion molecules on the sur&ace o& the endothelialcells3. Adhesion o& leu%ocytes leads to transmigration into the lowercham"er. he etent o& the transmigration can "e determined "y lysingthe cells and uanti&ying the &luorescence or "y counting the&luorescent cells3
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Methods &or la"eling o& cellular com/onents
8adioacti,e la"eling: e.g. with radioacti,e amino acids 45S7Methionin;3* which are incor/orated into newly synthesied/roteins. ulsechase e/eriments can gi,e insights into hal& li&e*trans/ort. rocessing; o& /roteins.
Internalisation o& high molecular weight mar%ers loading o&endosomes and lysosomes3.
$a"eling o& cell sur&ace /roteins e.g. "y "iotinylation with cell7im/ermea"le reacti,e "iotin com/ounds3.
Addition o& cell7/ermea"le la"eled su"stances* which integrate intos/eci&ic structures e.g. (olgi7s/eci&ic &luorescent li/ids3
rans&ection o& cells with e/ression /lasmids
rotein7ransduction cell7/ermea"le /e/tides3
Micro7In!ection o& su"stances.
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Amino acids: 45STMethionine* 4ET$eucin
Monosaccharides: 4ETMannose* 4ET(lucosamine
hos/hate: 42T /hos/horic acid Alternati,e: 44: lower radioacti,ity
Sul&ate: 45STSul&uric acid
atty acids: 4ETalmitinic acid
8adioacti,e $a"eling o& Biomolecules
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8adionuclides
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usually 45STMethionine la"eling
ad,antage: high s/eci&ic radioacti,ity
disad,antage: relati,ely rare amino acid chec% &irst*how many methionine are in the /roteinU e.g.+"iuitin: only 1 Methionine* "ut @ $eucines3
reuires Methionine7&ree culture medium &or de/letiono& endogenous stores "y /re7incu"ation
addition o& dialyed CS3
Meta"olic la"eling with amino acids
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4ETMannose or 4ET(lucosamine
Acti,ated in the cytosol "y cou/ling to nucleotides →
(#7Mannose* +#797Acetyl7(lucosamine +#7(lc9Ac3
reuires (lucose7/oor culture medium
Addition o& alternati,e energy sources
(lutamine* yru,ate3
Meta"olic $a"eling with Monosaccharides
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/reincu"ation without mar%er de/letion o&endogenous stores3
Addition o& the la"eled su"stance → VulseV 5 min 71 h or longer3
Sto/ o& the VulseV "y addition o& an ecess o&unla"eled com/ound
&urther incu"ation → VChaseV min 7 6? h3
Sto/ o& culture → Analyses
ulse7Chase7>/eriments
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i"ro"lasts o& /atients* were la"eled with 45S7methionine:
ulse: 1 h at 4<°CChase: < h at 1@°C and 4<°C 1@°C inhi"its trans/ort &rom trans7(olgi tolate endosomes/re7lysosomes3 Immuno/rec. and autoradiogra/hy
>am/le &or a ulse7Chase7>/eriment
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direct: modi&ication o& yrosine with 125JT:7 chemical oidation: Chloramine7 oidies 9a125Jand leads to iodination o& yrosine7 enymatic: $acto/eroidase: oidies 9a125J in/resence o& E2-2.
indirect: Modi&ication o& amino grou/s $ysine* 97erminus3 with 125JT7la"eled Bolton7Eunter78eagen
97succinimidyl 4767hydroy 57125
JTiodo/henyl37/ro/ionate3
Iodination o& roteins $a"eling with 125J3
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Eigh a&&inity "inding /artner o& A,idin andStre/ta,idin easy /uri&ication anddetection "y "eads* coated >$ISA7lates;3
Stre/ta,idin has 6 "inding sites &or "iotincom/lees can "e &ormed with "i,alent"iotin7lin%ersU3 signal am/li&ication is
/ossi"le ABC: a,idin7"iotin7com/lees3
Most commonly used method to la"el cellsur&ace /roteins
Biotinylation o& amino grou/s with Biotin7Eydroysuccinimid7>sters or o& cysteinesSE7grou/3 "y Maleimide7deri,ates
Biotinylation
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$a"eling o& >ndosomes* $ysosomes
By high molecular weight com/ounds* which are easilydetecta"le* not /ermea"le &or the cyto/lasmic mem"rane andta%en u/ "y endocytosis>am/les:a3 uns/eci&ic internaliation:7 IC7#etran luorescein7la"eled37 >nymes eroidase;3"3 S/eci&ically "y rece/tors
ulseChase conditions can "e used to la"eled s/eci&ically earlyor late endosomes or lysosomes e.g. using tem/erature"loc%s....3
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rans&ections
+sually designates the incor/oration o& #9A into mammalian cells. #9A /resentin &orm o& /lasmids.
ransient rans&ection: /lasmid remains outside o& the genome and is slowlylost degradation* dilution "y cell di,ision3* ece/tion: e/isomal re/lication De.g. S607lasmids in C-S7cells3. he trans&ection e&&iciency ,aries D "ut canreach close to 100P
Sta"le rans&ection: integration o& &oreign #9A into the genome >&&iciency:
usually "elow 0.1P3. Isolation o& sta"ly trans&ected clones reuires selectiongenes &or anti"iotic resistance* e.g. /uromycin* (61?;3. lasmids are usually
linearied "e&ore trans&ection to increase the /ossi"ility o& correct integration.
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>am/le &or a mammalian e/ression /lasmid
CM7romoter
Multi/leCloning Site
target gene
/oly7adenylation signalsuited &or lineariation
S607romoter
selction gene"acterialselection withKanamycin*mammalian7selection with(61?3
/oly7adenylation signal
8e/lication origins no shown3
re/orter gene
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Selection mar%ers &or sta"le trans&ections
Aminoglycoside7hos/hotrans&erase: 8esistance against 9eomycin"acteria3 und (61? mammalian cells3. Sele%tion with (61? ta%esuite long a// 2 wee%s3. Sur,i,ing colonies are isolated and &urthercultures under selection /ressure.
Eygromycin7hos/hotrans&erase
uromycin7Acetyltrans&erase
#ihydro&olat78eductase #E83: Selection with MethotreatF allowsam/li&ications o& the target gene.
(7usion /roteins: &luorescencecan "e used &or selection: eam/le:>(797κ B e/ressing CE-7cells
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Chemical rans&ection Methods
"#A-Calcium precipitates: at eact /E andCa2G7concentration: Eigh e&&iciencies with2@47cells @0P and more3* e/ression le,elsare usually moderate.
$iposome mediated transfection: %ationicli/ids which "ind the negati,ely charged #9A*and which are ta%en u/ as li/osomesWuite high trans&ection e&&iciencies "ut alsosometimes toic e&&ects and /otentially
arte&acts "y high e/ression le,els oralteration o& cellular mem"ranes. e.g.$i/o&ectamine* ugene...3
Ca2G
Ca2G
Ca2G
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Buffers
• HeBS-Buffer (Hepes buffered saline)
8 g NaCl - 280 mM final concentration
0.2 g Na2HPO4.7H2O (or 0.107 g anhydrous) - 1.5 mM
6.5 g Hepes (Sigma H-7006) (or 5.96 g of free acid) - 50 mM
400 ml A.dest.
Adjust the pH to exactly 7.05 (calibrate pH-meter with pH 4.01 and pH 7.00 buffers before). Add A.dest. to 500 ml,
filter through 0.2 µm filters and store in aliquots at -20°C (not longer than 6 months). Thawed aliquots shouldn't be
frozen again.
• CaCl2: 29.4 g CaCl2.2H2O (MW=147) in 100 ml A.dest (final conc.: 2 M) Filter through 0.2 µm filters and store
aliquoted at -20°C.
• Chloroquine (optional): chloroquine. 2H2O (Sigma C-6628): 12.9 mg/ml in PBS (conc.: 25 mM). Filter through
0.2 µm filters and store at -20°C.
Procedure (amounts are given for 6-wells):
1. Seed cells (about 500 000 cells per 6-well = per 10 cm2 ) one day before the transfection (in DMEM/10% FCS)2. (Optional: 1 h before transfection, exchange the medium for medium containing 25µM chloroquine)
3. Thaw HeBS and CaCl2 at room temperature
4. For each transfection prepare aliquots of 71 µl HeBS
5. Prepare the DNA/ CaCl2-Mix: 4 µg DNA (total) in 62 µl A.dest. + 9 µl CaCl2
6. Add the DNA/ CaCl2-Mix drop-wise to the HeBS aliquots (by screwing the Gilson pipette) and slightly mix after
each drop. Incubate for 2 - 3 min at R.T. to form the DNA-precipitate (not longer).
7. Add the DNA-precipitate drop-wise to the cells (by screwing the Gilson pipette and moving it to cover the whole
surface of the cell culture; don't swirl the dish).
8. Carefully transfer the dish back to the incubator. Incubate for 24 h (or in the presence of chloroquine: for 10 h)
and exchange the medium afterwards. (The transfection is in the presence of FCS!). The efficiency of transfection is
in the range of 70-90% for 293 cells. Harvest the cells after 48 h. The protocol is adapted from Neil Perkins who
adapted it from Gary Nolan in 1995
(See web site: http://www.stanford.edu/group/nolan/ or CP in Mol.Biol. 9.1 and 9.11.2-3)
rotocoll: Calcium7rans&ection
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46
$i/o&ectamine2000 7 Standard conditions
rans&ection in /resence o& serum e.g. 26h D 6?h3
47
Chemical rans&ection Methodes II
"endrimeres: charged olymeres that "ind#9A e.g. Su/er&ect Wiagen3
com/lees with "EAE-"extran /olycationic
#etran3
poly-et%ylene-imine &E'(:
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hysical rans&ection Methods
>lectro/oration: common withsus/ension cells*reuires much #9A S/ecial case: Amaa79ucleo&ection now $ona3
article7gun (ene (unX3:#9A on gold /articles shot "y/ressure onto cells e.g.neurons in "rain slices3.
Micro7In!ection o& #9A: intosingle cells !ust limited cellnum"er can "e targeted3.
electro7cu,ette
49
iral rans&ection Methods
Adeno)iruses* !etro)iruses*$enti)iruses:
iruses de,elo/ed &ancy mechanisms toget into cells D these are a//lied &orgene trans&er. irus7constructs aregenerated* which contain the target gene"ut not genes &or ,irus re/lication genes
essential &or generating the ,iral /articlesare su//lied "y ac%aging CellsX3.
Adeno,iruses: transient >/ression
8etro,iruses: sta"le integration "uttarget !ust /roli&erating cells
$enti,iruses: sta"le integration* alsotransduce uiescent* non7/roli&eratingcells.
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Adenovirus Retrovirus
•Episomal gene expression •Long-term, stable gene expression; inheritable
•Infects dividing &nondividing cells
•Infects dividing cells only
•High-level proteinexpression
•Moderate protein expression
•iral titers of !p to "#12
pf!$ml
•iral titers of !p to "#6 cf!$ml %an be
concentrated to "#9 cf!$ml'
•(ccommodates inserts of !pto ) *b
•(ccommodates inserts of !p to + *b
•Elicits imm!ne reactions in
vivo
•.oes not elicit imm!ne reactions in vivo
51
>am/le o& andadeno,iral system
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Adeno7associated ,irus AA3
is a small ,irus which in&ects human cells
is not causing any o",ious disease
causes a ,ery mild immune res/onse
can in&ect "oth di,iding and uiescent cells
AA ,ectors /ersists mostly in an etrachromosomal statewithout integrating into the genome o& the host cell the nati,e,irus can integrate to some etent into the host genome3.
romising gene thera/y ,ectors clinical trials ha,e "een done&or C8* hemo/hilia B* arthritis ;3
52
53
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54
ackaging cell line: %oenix cells. T%ese cells must not +e too confluent since t%is ,ill lo,er t%eproduction of )irus significantly.
"ay 1
late p%oenix cells on 1 cm plate &1.-1. x 1/ cells(
"ay 2
&&Transfect p%oenix cells using 2µ
g "#A and µ
l 0ugene 1.3 ml Opti4E4.'ncu+ate 5/ min !T and add to cells.(( O! +etter use CaO6777
"ay 5
Carefully remo)e old medium and add 2 ml of fres% medium.
'ncu+ate at 52°
C for 26% or 63% &or 2%(.
"ay 6-
8ar)est supernatant after 26-63%. 9irus sup can +e %ar)ested until t%e cells start looking un%ealt%y.
ut supernatant in / ml tu+e in ice +ucket in t%e %ood. Add 22 ml fres% medium to t%e packagingcells and put +ack into incu+ator.
Spin )iral sup to pellet any remaining cells.
0or storage of )irus: transfer to cryotu+e and snap freee ,it% #2 store at -3/°
C. ;pon freeing )irustiter goes do,n roug%ly t,ofold70or use straig%t a,ay: 0ilter )irus sup t%roug% a /.6-
µ
m cellulose acetate or polysulfonic filter&do #OT use nitrocellulose filter since it +inds proteins in t%e retro)iral mem+rane7(. <eep onice until use.
rotocol:roduction o& retro,irus "y trans&ection o& /ac%aging cells
55
#ay 4
S/lit the target cells into 10 cm dishes.
#ay 6
Add ? µgml /oly"rene to the &iltered ,iral su/ernatant. Mi gently "yin,ersion.
8e/lace the media in the target cells with the ,iral su/ G /oly"rene.Incu"ate at 4<°C &or h and add eual amount o& media containing/oly"rene.
#ay 5
8e/lace the media.
#ay
8e/lace the media with &resh media containing /yromycin to select &ortrans&ectants. he amount o& /yromycin used is determined "y %illing cur,ee/eriments. Select colonies o& sta"le trans&ectants %ee/ing the /yromycinin the media.
rotocol II:In&ection o& M>s with recom"inant retro,irus &or
ma%ing sta"le cell lines
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56
8e/orter7(ene Assays
E=0 >nhanced (reen luorescent rotein3 and ,ariants thereo& e.g.desta"ilied >(3
$uciferase
CAT Chloram/henicol7Acetyl7rans&erase3
>-=alactosidase &lac?(
SEA secreted al%aline /hos/hatase3 etc.
>nymes or other molecules* which are easily detecta"le* area//lied as Hre/orter molecules to detect /romoter acti,ities or theacti,ities o& signaling /athways.
he re/orter gene is cloned into an a//ro/riate /lasmid e.g.mammalian e/ression ,ector3 and trans&ected into the cells o&interest* &ollowed "y the "iological e/eriment e.g. stimulation3.
>am/les &or re/orter7genes:
57
8egulated romoters in 8e/orter (ene Assaysarti&icial /romoters3
andem 8e/eats o& transcri/tion &actor "inding sites e.g. 5 97%a//aB3. -&ten commercially a,aila"le e.g. &rom Stratagene or Clontech3 can "e used to determine the acti,ation o& a certain transcri/tion&actor or signaling /athway3
8e/orter (eneminimal/romoterelement
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8egulated* 9atural romoters in 8e/orter (ene
Assays
9atural /romoters usually contain "inding sites &or se,eraldi&&erent transcri/tion &actors sometimes se,eral co/ies o& asingle "inding site3 are regulated "y se,eral signaling/athways.>am/le:
IRF-1 GRE AP-1 NF/IL-6NF
κ
B
p65 / c-Rel
IL-8
Promoter
can "e used to determine the regulation o& a s/eci&ic /romoter o& interest$uci&erase is usually easier to measure with high sensiti,ity than the gene o&interest in this case I$7?3
$uci&erase
59
9ormaliation constructs with constituti,e /romoters
C49 : &rom human Cytomegalo7irus: induces strong constituti,ee/ression &ast* a"out 1 day a&ter trans&ection3
!S9 : &rom 8ous Sar%oma7irus: wea%er* "ut ,ery constant* constituti,ee/ression* slightly slower ta%es 2 d3
S96/: Simian irus 607romoter
Actin-romoter: human /romoter o& a Hhouse%ee/ing gene
;+i@uitin-romoter: human /romoter o& a Hhouse%ee/ing gene
;usually used &or the normaliation control in re/orter gene assaysto com/ensate &or di&&erences in trans&ection e&&iciency* etraction e&&iciencyor ,ia"ility o& cells in di&&erent sam/les3
+sual setting: athway7s/eci&ic re/orter construct e.g. with &ire&ly luci&erase3G constituti,e normaliation construct e.g. +"iuitin7romoter dri,en β7galactosidase or 8enilla7luci&erase3 uanti&ication o& normalied ,alues
$uci&erase β7galactosidase or ire&ly 8enilla7luci&erase3
romoters for t%e normaliation )ector:
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>am/le o& an 8e/orter (ene Assay
60
46.36
0.90 0.491.94
26.66
4.63
0
5
10
15
20
25
30
35
40
45
50
p53 Rb E2F
f o l d o
f c o n
t r o l v e c
t o r
EP-cells
DU145
Acti,ities o& di&&erent signaling /athways or molecules /54* 8"* >23 areassessed with re/orter constructs containing res/ecti,e transcri/tion&actor "inding sites u/stream o& a luci&erase ,ector.
or normaliation /ur/oses a constituti,ely e/ressed control gene hasto "e cotrans&ected e.g. β7galactosidase downstream o& a constituti,e/romoter e.g. u"iuitin7/romoter3. alues are calculated as $uci&eraseβ7(al.
$ u c i & e r a s e β 7 ( a l
61
8e/orter7(ene Assay Systems &or Analyses o&Signaling athways
(ene o& interest
8e/orter7lasmid
ranscri/tion &actor construct
In case that the gene o& interestsacti,ates the s/eci&ic /athway*which leads to /hos/horylationand acti,ation o& the usedtranscri/tion&actor construct* thee/ression o& the re/orter e.g.luci&erase3 is induced.
ranscri/tion &actor construct
8e/orter7lasmid
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#etection o& re/orter genes: ( and ,ariants3
luorescence measurement "y&luorometry e.g. in @7well&luorescence readers3
Microsco/y
low analysis cytometry*ACS: luorescence acti,atedcell sorting: (7containingcells can "e se/arated &romother cells and i& necessaryalso &urther culti,ated a&ter/uri&ication3
( &luorescence
63
#etection o& $uci&erase ire&ly or 8enilla $uci&erase3
PMTLuciferase-
Zellextrakt
ATP
Luciferin
M: &rom htt/:www.moleculare/ressions.com
ery sensiti,e detection in cell etracts "y measuring theluminescence generated &rom luci&erase in /resence o&luci&erin and A. he su"strate $uci&erin has to "e in!ectedinto the sam/le* and measured immediately e.g. "yintegrating &or 5 sec3 as the emitted luminescence decaysuic%ly. >mitted /hotons are measured with /hotomulti/liertu"es.Measuring de,ices: $uminometeralso as @well de,ices a,aila"le3
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64
#etection o& β7(alactosidase lac)3
o&ten "y /hotometry e.g. in >$ISAreaders3 using a yellow su"strates* whichturns red in /resence o& (alactosidasesu"strate: C8(* Chloro/henolred7β7#7(alacto/yranoside3. #etection at 5@5 nm.
Alternati,e: Chemiluminescencemeasurement
Alternati,e: luorimetric #etection:Su"strate: e.g. 47car"oyum"elli&eryl"eta7#7galacto/yranoside* C+( iscon,erted to a &luorescent /roduct
Microsco/ical detection: with Y7(al 57Bromo767chloro747indolyl7Z7#7galactoside3as su"strate: /roduces a dar% "luereaction /roduct
65
$yse cellsrecommended cell lysis "u&&er: 0.25M risECl /E ?.0* 0.25P ,,3 960*2.5 mM >#A3
i/et a"out 10 Ql o& etract into a @7well /late a//ro/riate neg. control "lan%: 10 Ql o& moc%trans&ected cells* or non7trans&ected cells D as there is a slight endogenous "7(al acti,ity3 D lea,e onewell em/ty &or "lan% A713
Add 100 Ql su"strate solution to the wells also to the "lan%7well3
Incu"ate until red color de,elo/s min to hours D de/ending on "7(al acti,ity* i& you ha,e low acti,ityyou can also incu"ate at 4<°C3
-/tional: Sto/ with 50 Ql o& Sto/ solution only necessary i& you want to time it eactly* e.g. "y adding
the su"strate in a timed way and sto//ing the reaction in the same way3
Measure with >$ISA 8eader at 5<0 nm ilter [43 o/timum: 5@5 nm
$ysis Bu&&er:
0.25M risECl /E <.6 or "etter ?.03
0.25P ,,3 960
2.5 mM >#A
C8(7su"strate solution: 1 mgml \ 1.5 mM3
in BS G 10 mM KCl* G 1 mM MgCl2
alternati,e su"strate "u&&er:
0 mM 9a2E-6 /E ?.0* 1 mM MgCl2* 10 mM KCl* 50 mM Merca/to7ethanol
Sto/ solution: 0.5M 9a2C-4
β7(alactosidase Assay with C8(
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66
Methods to su//ress gene e/ression89A inter&erence3
Antisense7echnologies: to su//ress gene e/ression were started many yearsago. >arly a//roaches used antisense7oligonucleotides D "ut the e&&ect was ,ery,aria"le.
Alternati,e a//roaches used long antisense7strands hy"ridiing to the m89A:his usually leads to downregulation o& gene e/ression D "ut uite o&ten notonly &or the targeted gene D "ut also uns/eci&ically &or other genes. he reasonis that this is Hsensed "y the cells li%e a long ,iral ds89A* leading to ,irusde&ense mechanisms: acti,ation o& K8 /rotein %inase 83* /hos/horylation o&translation &actors and general downregulation o& /rotein synthesis.
Some years ago* scientists &ound that small ds89A in the range o& 1@721nucleotides inter&eres s/eci&ically with target genes* without a&&ecting othergenes small inter&erent 89A* si89A3 D "ecause they are too small to acti,ate,irus de&ense mechanisms.
67
rinci/le o& 89A7inter&erence
Small ds89A si89A3 "inds to an89A7induced silencing com/le8ISC3F consisting o& argonaute/roteins.
ogether with the 8ISC* one 89Astrand "inds to the target m89Aand leads to s/eci&ic degradationo& this m89A.
he 8ISC com/le is Hrecycledleading to degradation o& additionm89A7targets.
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micro789AOs D the "iological mechanism to
su//ress gene e/ression
mi89As D can ha,etwo e&&ects:
13 m89A degradation
23 inhi"ition o&translation thise&&ect o&ten doesnot reuire a
100P match withthe target m89AU3
69
Chemically synthesied si89A
htt/:www.dharmacon.com
htt/:www.mwg"iotech.comhtmls]synthetic]acidss]rna.shtml
he two short 89As are annealed and trans&ected usually usingmethods that are suited &or short oligonucleotides e.g.$i/o&ectamine2000 &rom In,itrogen* Ytreme(ene &rom 8oche;3.
#esign: traditionally the &irst AA7#u/lett is searched D and the &ollowing 1@"ases are chec%ed &or (C7content should "e 60 D 50P3* the seuenceshould "e target gene s/eci&ic chec%ed "y B$AS3 D an a//ro/riate 89Aseuence and the re,erse com/lementary 89A are chemically synthesiedand ordered "y a com/any* e.g. #harmacon* In,itrogen* ML(*;3.
Com/any home/ages o&ten o&&er a "asic si89A design:
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ector7coded si89A small hair/in 89A sh89A3
the normal trans&ection methods* o/timied &or /lasmids can "e used
Anti"iotics selection genes e.g. (61?* /uromycin;3 can "e included sta"le H%noc%7down cell lines can "e generated www.imgene.com*htt/:www.oligoengine.com3
71
small hair/in 89A: hair/in7loo/
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72
ro&essional #esign o& si89A or sh89A
#esign ,ia com/any we"sitehtt/:www.thermoscienti&ic"io.comdesign7center^redirect\truehis deli,ers a list o& se,eral /ossi"le seuences gene s/eci&ic*chec%ed "y B$AS3 D with a score "ased on em/irically determinedcriteria: 9ature Biotechnology 22* 427440* 2006
Chec% literature &or &unctional si89A seuences
or transduction o& /rimary cells: lenti,iral sh89A constructsalso wor% in non di,iding cells3
there are also induci"le lenti,iral constructs a,aila"lehtt/:tronola".e/&l.ch3
Many ,ectors can also "e o"tained &rom /lasmid re/ositories: Addgene: htt/:www.addgene.org
Belgian re/ository: htt/:"ccm."els/o."ed"lm"/]search]&orm./h/
(ene re/lacement strategy
73
gene o& interest5O+8 4O+8
si89A targeting the endogenous
m89A ,ia theuntranslated region
mutated gene o& interest
&oreign4O+8
S60olyA3
the mutated genere/laces the endogenousgene
good/romoter
>/ression /lasmid containing:
endogenous m89A
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Im/ortant controls in si89A e/eriments
scram"led si89A as negati,e control
mutated si89A with some mismatch as negati,e control note:might act as mi89A U3
other si89As targeting the same m89A should ha,e the samee&&ect
i& you use sh89A ,ector "ased 89A7inter&erence3 use anunrelated sh89A as negati,e control e.g. sh89A ,ector withscram"led si89A seuence3.he em/ty sh89A ,ector is not a ,alid negati,e control
75
Micro-Injection:
This allows injecting antibodies
against certain endogenous
proteins > interfering with their
functions. However, just a limited
number of cells (e.g. up tohundreds with automated systems)
can be targeted > the following
analysis should be a single cell-
based assay, such as microscopy.
Alternati,e Methods to in&luence endogenous/rotein le,els
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76
8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions
&luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
77
Analysis o& roteins "y S#S7A(>
S7S
S-67
7
77
77 777
7 7 77
7777
77
reducing agent #3or Merca/to7>thanol7 "rea%s disul&ide
"onds@5°C
S#S D sodium dodecyl
sul&ate: coats /roteinswith negati,e charges
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78
S#S7(els
For final concentration of gel ( % T):
Separating gel (10 ml)
Stack gel
(10 ml)
7% 10% 12,5 % 15% 5%
30% Acrylamide-bissolution 29:1(A) 2.33 3.33 4.17 5 1.67
4x Separation buffer
2.5 2.5 2.5 2.51.5 M Tris/HCl pH
8.8
4x Stacking buffer
2.5
0.5 M Tris/HCl pH6.8 + phenolred
aqua dest. 5 4 3.2 2.4 5.7
SDS (10 %) 0.1 0.1 0.1 0.1 0.1TEMED 0.015 0.015 0.015 0.015 0.015
APS (10%) 0.03 0.03 0.03 0.03 0.03
2 M Tris-Cl
(pH 6.8)
2.4 ml
SDS 0.96 g
Glycerol 4.8 ml
DTT 739 mg
Bromophenol
Blue4.8 mg
S#S7"u&&er
79
Coomassie7Blue staining: ro"ust*moderate sensiti,ity limit ≈ 1 Qg3
Sil,er staining: elementary sil,er isde/osited at the site o& /roteins*,ery sensiti,e limit ≈ 10 ng3
/rotein7s/eci&ic &luorescent dyes:S=8-7-range* S=8-78u"y*#ee/7ur/lecom/ati"le with MA$#I7-* MS3
S/ecial stainings: roteoglycansAlcian7Blau3* glyco/roteinsSchi&&Os 8eagent3
Autoradiogra/hie* luorogra/hy
#etection techniues
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80
k"a
2 k"a
se/aration with 12P Acrylamide: 20 7 ?0 %#a
Sil,er staining
Molecular weight assessment a&ter S#S7A(>
81
htt/:www.meduniwien.ac.atuser!ohannes.schmidS#S7A(>.ls
logMLT
migration distance starting &romstac%ing gelse/aration gel inter&ace
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82
Sensiti,e detection o& radioacti,ely la"eled /roteins
(el is euili"rated with a radiosensiti,e &luoro/hore:e.g. #i/henyloaole -3* Sodiumsalicylate
#etection "y Y7ray &ilm or hoshor7Imager de,ices
luorogra/hy* Autoradiogra/hy
83
hos/ho7Imager detection1 day e/osure3
luorogra/hy
Y7ray &ilm detection4 months e/osure UU3
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84
Solutionsiing solution: 50 P ethanol* 10 P glacial acetic acid* ad 100 P with aua dest.Incu"ating solution 1$3: 40 P ethanol* sodiumthiosul&ate anhydrous 2g* sodiumacetat
anhydrous 46 g* &ill u/ to 1$ with aua dest. Be&ore use add 125 Q$ o&glutaraldehyde50 m$ incu"ating solution.
Sil,ernitrate solution 1$3: Ag9-4 1 g* dissol,ed in 1$ aua dest.. Be&ore use add 10 Q$o& &ormaldehyde50 m$ o& sil,er nitrate solution.
#e,elo/ing solution 1$3: 9a2C-4 anhydrous 25 g* dissol,ed in 1$ aua dest.. Be&oreuse add 10 Q$ o& &ormaldehyde50 m$ o& de,elo/ing solution.
Sto/ solution 1$3: sodium7>#A 15.<? g dissol,ed in 1$ aua dest..
A&ter electro/horesis* the /olyacrylamide gel is ta%en out o& the casting sandwich and/laced in a clean glass "ea%er &illed with &iing solution. All &ollowing ste/s arecarried out while gently sha%ing. he gel has to "e incu"ated with the &iingsolution &or 40 minutes. A&ter &iation an a//ro/riate amount o& incu"ating solutionincluding glutaraldehyde the gel has to "e at least co,ered "y liuid3 is /re/ared
and added to the gel* &ollowed "y incu"ation &or 15 minutes* discarding the &iingsolution and washing with aua dest. 4 &or 5 minutes and 10 minutes incu"ationin sil,ernitrate solution including &ormaldehyde. he sil,ernitrate solution iscollected s/ecial waste3. #e,elo/ing is carried out "y incu"ating the gel inde,elo/ing solution including &ormaldehyde until the desired intensity o& /roteinstaining is reached* &ollowed "y discarding o& de,elo/ing solution and adding sto/solution. he gel should incu"ate &or at least 1 hour in the sto/ solution. A&terwards the gel can "e stored in aua dest. or dried with ,acuum.
Sil,er Staining o& A(> (els
>MSAOs >lectro/horetic mo"ility shi&t assays3
;used to monitor acti,e transcri/tion &actors "y "inding to short* la"eledoligonucleotides com/rising the "ound #9A seuence3
>am/le: com/.: com/etitor: non7la"eledds7oligo o& the same seuenceusually added in 107&oldmolar ecess3 D com/etes withthe la"eled oligo &or "inding tothe reduces the s/eci&icsignal
mut.com/.: mutated com/etitor:should not com/ete &or s/eci&ic"inding
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#e&ining the com/ositiono& 7com/lees using
anti"odies and su/ershi&ts
su/ershi&t
>MSA Alternati,e: ABC# AssayA,idin7Biotin Com/le with #9A3
87
Stre/ta,idin
Biotin
ds-ligo
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88
Se/aration o& /roteinsaccording to their isolelectric/oint /E at which they arenot charged3
+sage o& immo"ilied /E7gradients Am/holines3
nati,e I> or denaturing I>urea3 can "e done
Isoelectrical ocussing I>3
89
&or analyses o& com/le /rotein mitures
→ roteomics3
com"ination o& I> 1. dimension3 and S#S7A(> 2.dimension3
high se/arating resolution > 1000 S/ots3 rotein /attern data"ases are a,aila"le and so&tware
&or /attern com/arison
ident&ication o& s/ots "y mass s/ectrometry or "yimmunological methods immuno"lotting3
2#7>lectro/horesis
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90
rinci/le o& 2#7>lectro/horesis
91
2#7S#S7A(>
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2#7#I(> #i&&erence (el >lectro/horesis3
rotein etracts &romtwo di&&erent sam/lesare la"eled with twodi&&erent &luorescentdyes e.g. Cy2 and Cy43and mied usuallytogether with a /ooledstandard miturela"eled with a 4rd dye*eg. Cy53.
2#7A(> is /er&ormed*&ollowed "y scanning o&the gel with the 4wa,elengths ecitingthe 4 dyes the resultsare com/ared "ycom/uter analysis
(el chromatogra/hy sie7eclusion chrom.3
Se/aration techniue that uses /orous "ead material in acolumn to se/arate macromolecules according to sie.
Lide range o& molecular weights that can "e se/arated
Much larger molecules can "e se/arated than with S#S7A(> ornati,e A(>
93
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(el chromatogra/hy
94
larger molecules are eluted &irst
(el chromatogra/hy
95
0.001
0.003
0.005
0.007
0.009
0.011
0.013
0.015
0.017
0 5 1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
1 0 5
1 1 0
min
E x
4 3 6 / E m
5 1 0
-0.01
0.01
0.03
0.05
0.07
0.09
0.11
0.13
0.15
0.17
0.19
T r p - f l u
o r e s c e n c e
SERT
Standards
670 kDa
158 kDa
44 kDaSERT
>am/le
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S/in desalting or "u&&er echange "ased on gelchromatogra/hy
1. lace s/in column in a 1.5 m$ microcentri&uge collection tu"e.
2. Centri&uge at 1500 g &or 1 minute to remo,e storage solution.
4. Add 400 _$ o& desired &inal "u&&er to the resin "ed and centri&uge at 1500 g &or 1 minuteFdiscard &low7through
6. lace the euili"rated s/in column into a new collection tu"e
5. $oad your /rotein sam/le 140 Ql3
. Centri&uge at 1500 g &or 2 minutes to collect desalted sam/le.
<. rotein sam/le is now desalted ` "u&&er echanged and ready &or use
96
97
Lestern Blotting: >lectro/horetic /rotein7trans&er&rom a gel mostly S#S7A(>3 onto a mem"rane
Mem"ranes: 9itrocellulose*oly,inyl7di&luorid #3
detection ,ia enyme7cou/led anti"odies
>nymes: 7 Eorse 8adish eroidase E83
7 al%aline /hos/hatase A3
Immuno"lotting
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98
Immuno"lotting II
99
Let Blottingeui/ment
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100
Blotting conditions
101
Semidry "lotting Bio8ad3
wet Milli/ore Immo"ilone7mem"rane in Me-E* rinse withwater* then 1 min. in Blotting "u&&er:25mM ris Base150 mM glycine10P methanolma%e a @0P stoc% without Me-E*
which is then added &reshly3&ilter 0.2 mm UUU wet 5 4MM &ilter /a/ers 7 anode then wetted mem"rane then gel then 5 wetted &ilter /a/ers wet cathode* close lid uic%ly
without mo,ing "ac% and &orth. "lot 250 mA &or a small Bio78ad gel
should "e 20* increasing to 40 3&or 40 min.
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102
Chromogenic
detection o& E8
Immuno"lotting: ,arious su"strates
Chemiluminescence
7su"strate
103
Lestern Blot rotocol
"etection: "loc% 1 hour to -9 in BS5P mil% at 8 wash 4 BS0.1Pween 20 1st AB in BSween* 1 h wash 4 5 min BS0.1Pween 20
add 2nd AB in BSween e.g.* /eroidase7con!ugated don%eyanti7ra""it Amersham* at 1:10.0003 &or 1 hour
wash 4 5 min
EC$-system: com"ine eual ,olumes o& soln. A and soln. B Amersham >C$ or
ierce Su/erSignal Lestico3 add to &ilter* incu"ate 1 min. Amersham3* or 5 min ierce3 roll away* co,er with saran wra/* e/ose within 10 min. e/osure times can ,ary "etween seconds and 20 min.3
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104
Ad,antages: almost no costF sta"lere/roduci"le* stored in &roen aliuots
o 10 mls o& 100 mM ris /E ?.5 83* add
50 Ql luminol warm to redissol,e3
22 Ql o& coumaric acid warm to redissol,e3
4 Ql o& E2-2 &resh months3
our onto "lot &or 1 minute and /rocess as normal 10 ml enough &or 100cm23
Stock luminol: 250 mM 47amino/thalhydraide lu%a [0@2543F 2mgs in mls #MS-F store &roen in 0 Ql aliuots.
Stock coumaric acid: @0 mM coumaric acid Sigma C@00?3: 4? mgs in2.5 mls #MS-F store &roen in 25 Ql aliuots.
ST!''#= of mem+ranes: with 2P S#S* 2.5mM 8IS /E.?*100mM Beta7merca/toethanol &or 40 min at 50°C
>C$ reagent: sel&made ,ersion
105
Interaction "etween anti"ody andantigen in solution
Isolation o& immuno7com/lees "yrotein A7 Agarose Se/harose3 D Beadsrotein A \ Ig(7"inding /rotein o& Sta/hylococcus aureus
Alternati,e: rotein (: o&ten used &ormouse monoclonal anti"odies* which are notwell "ound "y rotein A
7 or anti"ody co,alently lin%ed to acti,ated
Se/harose C9Br7acti,ated3
Analysis "y S#S7A(>
Immuno/reci/itation Antigen
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106
>am/le o& an Immuno/reci/itation
roteins la"eled uns/eci&ically with 45S7methionine s/eci&ic detection o& a/rotein o& interest "y immuno/reci/itation &luorogra/hy
A&&inity chromatogra/hy
"eads can "e cou/ledwith anti"odies or othera&&inity ligands* which"ind a molecule o&interest.
a&ter washing o& the"eads* the ca/turedtarget molecules can "eeluted e.g. "y lower /E.
107
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108
/roteolytic /rocessing
(lycosylation
hos/horylation
+"iuitination
;.
Analysis o& /ost7translationalmodi&ications
109
in ,i,o: culti,ation o& cells in /resence o& selecti,e/roteinase inhi"itors
inhi"ition o& intracellular /roteinase in cytosol* >8*
(olgi3 is /ossi"le !ust with mem"rane /ermea"leinhi"itors
inhi"itors that are not mem"rane /ermea"le act !ustin the etracellular en,ironment* in endosomes andlysosomes
in ,itro: Incu"ation with s/eci&ic /roteinases
Analysis o& /roteolytic /rocessing
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110)7A7CE92 .... Benyloycar"onyl7henylalanyl7Alanyl7
#iaomethane inhi"its cysteine /roteinases3
)7A7CE92
roteolytic /rocessing in ,i,o3
111
in ,i,o: Biosynthesis in /resence o& inhi"itors
unicamycin: inhi"its the initiation o& 97(lycosylation
rocessing o& 97(lycans "loc%ed "y (lycosidase7Inhi"itors
#etection o& 97(lycosylation
unicamycin µgml3 0 0.1 0.5
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112
>ndo E .... >ndoglucosaminidase EF clea,es !ustMannose7rich 97(lycans
9(ase ... e/tid:97(lycosidase V97(lycanaseV3
Analysis o& (lycosylation in ,itro3
113
in ,i,o: meta"olic la"elling with42Thos/hate e.g. in /resence ora"sence o& an e/ression or su//ressionconstruct &or a s/eci&ic %inase* &ollowed "yimmuno/reci/itation3
in ,itro:
7 Incu"ation with al%aline hos/hatase
7 hos/ho7aminoacid analysis #C3
7 Kinase7Assays
Immuno"lottingImmun/reci/itatione.g. with anti7/hos/ho7 s/eci&ic Anti"odyor &or instance anti7hos/hotyrosin3
Analysis o& hos/horylation
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114
9(ase ... e/tid:97(ly%osidase V97(ly%anaseV3
Al%hos ... Al%aline hos/hatase
In ,itro hos/horylation Analysis
115
Immuno/reci/itated Kinase
anti"ody
Agarose7Bead
su"strate /hos/horylated su"strate
427γ A G
1. Immuno/reci/itation o& the %inase
2. Kinase78eaction in /resence o& 427γ 7A
4. S#S7A(> and luorogra/hy
in ,itro Kinase Assay
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116
$ysis "u&&er &inal conc.3: &or 20 ml:
20 mM risECl /E<.5600 Ql 1 M150 mM 9aCl00 Ql 5 M25 mM "7glycero/hos/hate500 Ql 1 M2 mM >#A?0 Ql 0.5 M2 mM/yro/hos/hate600 Ql 0.1 M1 mM ortho,anadate200 Ql 0.1 M1P riton Y71002 ml 10P1 mM #20 Ql 1 M1 mM9a20 Ql 1 MA. dest.15.? ml
rotease Inhi"itors: added "e&ore use $eu/e/tin* e/statin* e&a7Bloc%3 according to stoc%
Kinase "u&&er &inal conc.3: &or 20 ml:20 mM risECl /E<.5600 Ql 1 M20 mM "7glycero/hos/hate600 Ql 1 M100 QM ortho,anadate20 Ql 0.1 M10 mMMgCl2200 Ql 1 M50 mM 9aCl200 Ql 5 M1 mM #20 Ql 1 M50 QM A50 Ql 20 mM1 mM 9a20 Ql 1 MA. dest.1?.<ml
$yse cells in wells3 with 500 Ql /er well o& $ysis "u&&er G /rotease inhi"itors3:20 min at 6°C.
Clear "y centri&ugation 16000 r/m* 6°C 15 min >//endor& centri&uge3. Sa,e an aliuot 40 Ql3 &or Lestern "lotting.
Immuno/reci/itate the %inase e.g. with 10 Ql anti7&lag a&&inity matri "eads* Sigma* &or &lag7tagged trans&ected %inaseF orwith a//ro/riate anti"ody &or endogenous %inase G rotein A7Se/harose or directly cou/led to agarose3: 2h at 6°Crotating3.
Lash the "eads: 4 with 1 ml BS 6°C3* 1 with 1 ml Kinase "u&&er 6°C3: /ellet the "eads "y centri&ugation 16000 r/m*
6°C* 65sec3 and remo,e the su/ernatant.re/are Kinase "u&&er: add MnCl2 to 10 mM stoc%: 1 M3 and 427g7A 5 QCi /er sam/le* usually 110 ,olume* i.e. 1 Ql
o& stoc% solution &or one 10 Ql assay3 and /reincu"ate at 40°C &or 10 min.
Add 1 Qg su"strate: (S7I%B 1 Ql3 or mutant su"strate as control3 to the "eadsF add 10 Ql %inase "u&&er* mi gently andincu"ate at 40°C &or 40 min or longer3.
Sto/ the reaction "y addition o& 6 S#S7sam/le "u&&er 6 Ql3 and /er&orm S#S7A(> with the sam/les* &ollowed "y &iationo& the gel 10P methanol* 10P EAc3* drying and autoradiogra/hy.
or detection with hast(el: use only 5 Ql "eads* 5 Ql %inase "u&&er* 0.5 Ql su"strate and 2 Ql 6 S#S7sam/le "u&&er: 8un a12.5P hast(el with 6 Ql /er sam/le
Kinase Assay7 rotocol
#etection o& +"iuitination
trans&ection o& a tagged u"iuitine.g. Eis7tagged3 together with thegene o& interest e.g. &lag7tagged3
immuno/reci/itation o& the geneo& interest
o/tional: resus/end and heat the
"eads in 1P S#S7"u&&er* dilute to0.05P S#S and re/eatimmuno/reci/itation to get rid o&/otential co7/reci/itating*interacting /roteins.
resus/end and heat the "eads inS#S7A(> "u&&er
S#S7A(>
Lestern Blot &or the Eis7tag
otential set7u/:
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118
most o&ten used: density gradient centri&ugation
coarse se/aration: di&&erential centri&ugation
#etection o& su"cellular com/artments "y s/eci&ic
mar%ers enymes that are nearly eclusi,ely in thatcom/artment3
Su"cellular ractionation
Se/aration o& su"cellular com/artments3
119
#i&&erential centri&ugation
Su"seuent centri&ugation ste/s with increasing g7&orce
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120
(radient centri&ugation:
Sam/les are usually layed on to/ o& a gradient* /roteinsor com/artments migrate through the gradient in Hones
121
(radients: continuous ↔ discontinuous Hste/sV3
sel&7&orming gradients e.g. ercoll3
density euili"rium centri&ugation3
sam/les are either layered on to/ o& the gradient or at the
"ottom
ractionation a&ter the centri&ugation e.g. "y /eristatic/um/ and &raction collector3
#ensity gradient centri&ugation
A B
A
B0
1000
100
,olume or time3Magnetic stirrer
gradient mixer:
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122
Sucrose: low molecular weight 462 #a3*
osmotically acti,e
icoll: co/olymerisate o& Sucrose and>/ichlorhydrineF Mr ≈ 600 000 #a
ercoll: colloidal silica gel
s/ecial case &or #9A: Cesiumchloride
Su"stances to generate density gradients
123
Sel&7&orming gradients e.g. ercoll..3
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124
Se/aration o& lysosomes 3 and (olgi 3
in a continuous ercoll gradient density b3
>am/le &or a density gradient centri&ugation
125
8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions &luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
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126
Methods to in,estigate macromolecular interactions
Interaction screening with /hages hage #is/lay3
=east 17Ey"rid System /rotein : #9A3
=east 27Ey"rid System /rotein : /rotein3
Mammalian 27Ey"rid System
(el7Chromatogra/hy
Co7Immuno/reci/itation
luorescence 8esonance >nergy rans&er 8>3 D see &luorescence methods
127
Interaction screening with /hageshage #is/lay3
A gene li"rary is e/ressed on thesur&ace o& a//ro/riate /hages e.g.M143* which are incu"ated with s/eci&ictarget /roteins immo"ilied on /lates.+n"ound /hages are washed o&&F "ound
/hages are eluted "y lowering the /E.3-4 x
Bound /hages are am/li&ied and againincu"ated with /lates containing the target/roteins D this re/eated 4 D 6 to enrich thes/eci&ically "inding /hages. Clones are isolatedand seuenced Seuence o& the "inding
/rotein
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128
=east 17Ey"rid System
#9A78egion with /otential /rotein"inding sites in re/eats3
AA EIS lac)
(al67Acti,ation domain
Insert &rom li"rary
ranscri/tion
or the identi&ication o& /roteins that "ind s/eci&ically to a gi,en #9A seuence e.g.transcri/tion &actorsF #9A:/rotein interaction3.
he #9A seuence o& interest e.g. &rom a /romoter3 is usually cloned in re/eats 4753in &ront o& an a//ro/riate selection gene e.g. a histidine synthesis gene3 and ana//ro/riate re/orter yeast strain which is not ca/a"le o& growing in the a"sence o&histine3 is sta"ly trans&ormed with this construct. Su"seuently* this yeast strain istrans&ormed with a li"rary containing /utati,e "inding /roteins o&ten &used to thetransacti,ation domain o& the (al6 transcri/tion &actor3. Binding o& a /rotein to the #9Aseuence results in growth o& this yeast clone on selection /lates.
growth onselection /lates
129
=east 27Ey"rid System
A yeast strain is used* which does not contain a &unctional (al6 transcri/tion &actor D"ut re/orter and selection genes* which are downstream o& (al67de/endent /romotersEistidine7 and Adenine7synthesis genes* lac) &or β7(alactosidase e/ression* whichcan "e used &or staining3. his strain is trans&ormed with /utati,e interaction /artners:
In case o& an interaction "etween /rotein Yand = au&tritt* a &unctional transcri/tion &actor is"uild* which "inds to (al6 /romoters D and thecells can grow on selection /lates without Eisor Ade3.
2. &usion /rotein o& the (al67transacti,ation domain and rotein = or ali"rary insertF \ H/rey3
1. &usion /rotein o& the (al67#9A7"indingdomain and rotein Y H"ait3
Co7rans&ormation or com"ination "yyeast mating
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130
re/aration o& a =east 27Ey"rid Screen
1. Cloning o& the gene o& interest into the "ait,ector in &rame with the (al67#9A7"inding domain3: selection in "acteria e.g.,ia Kanamycin3* selection in yeast e.g. ,iar/7synthesis gene33
2. est &or Auto7Acti,ation with Hem/ty(al6A#7,ector3: ests whether the gene o&interest interacts with the (al6 acti,ationdomain without the need o& /rotein =3:I& it does so* the "ait cannot "e used in theyeast 27hy"rid system.
he (al6 A# ,ector contains a second
selection gene &or yeast e.g. leucinesynthesis gene3. Co7trans&ormants o& thetwo ,ectors grow in the a"sence o& r/ and$euF "ut they should not grow in thea"sence o& the amino acid that issynthesied !ust when an interaction occurse.g. in the a"sence o& histidine oradenine3.
131
Com"ination o& the two /utati,e interaction /artners
X
PCR from single colonies (with primers specific for the library vector)
sequencing of the putativeinteraction partners
purification of PCR-Products
His, Ade,lacZ
Reporter strain Y187pretransformed with a library (inGal4AD-Vector), Mating Type: α
Reporter strain(e.g. AH109)
Mating Type: a
Mating: Incubation of the two haploidstrains for 24 h at 30°C, 40 rpm
> formation of diploid clones with bothvectorsclones, which contain interactionpartners grow on selection plates andexpress lacZ
Gal4ADX
Gal4BD
bait
Instead of mating the two vectors can becombined by classical transformation
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132
>am/le o& a yeast two7hy"rid result
A3 single colonies on selection/lates S#7$eu7r/7Ade3
B3 Strea%ing out the colonies &rom the&irst selection /lates to secondaryselection /lates e.g. with higherselection /ressure and stringency:S#7$eu7r/7Ade7Eis3
133
eri&ication o& a yeast 27hy"rid result
1. Analysis o& the seuence and com/arison with data"ase: chec% whether the -8is -K in &rame with the (al6A#3
2. Isolation o& lasmid7#9A &rom the yeast colony
4. 8e7trans&ormation in >.coli to se/arate "ait and /rey D using di&&erent anti"ioticsresistance3 and /re/aration o& the /lasmid containing the li"rary insert
6. Halse ositi,e est in yeast: rans&ormation o& the (al6A#7/lasmid containingthe identi&ied H/rey with the em/ty (al67"inding domain ,ector: this should notlead to growth on selection /lates o& interaction i& there is growth* then theli"rary insert interacts with the (al6B# and not the "ait /rotein3
5. β7(alactosidase7assays also uantitati,e* to com/are interaction /artners3
. eri&ication in the correct cells human cells3* e.g. "y co7immuno/reci/itation
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134
>am/le o& a alse7ositi,e estX
>am/le o& lac)uanti&ication
1
1,1
1,2
1,3
1,4
1,5
1,6
1,7
neg. control IKK2/ GMRa IKK2/GMRb
r e l . a c
t i v i t y
135
Solutions: Synthetic dro/ out solution 10 in A#: $7isoleucine 400 mg$* $7,aline 1.5 g$* $7adenine hemisul&ate salt 200
mg$* $7arginine ECl 200 mg$* $7histidin ECl monohydrate 200 mg$* $7leucine 1 g$* $7lysine ECl 400 mg$* $7methionine 200 mg$* $7/henylalanine 500 mg$* $7threonine 2 g$* $7try/to/han 200* $7tyrosine 400 mg$* $7uracil 200 mg$
S# 7r/ medium synthetic dro/out medium3: synthetic minimal medium lac%ing try/to/han: yeast nitrogen"ase without amino acids .< g$* 2 P detrose glucose3 sterile detrose solution is added a&ter autocla,ingto a,oid maillard reactions3* /E ad!usted to 5.?* &or /lates : agar 1.5 g$
=# yeast /e/tone detrose3 "roth* yeast com/lete medium: yeast etract 10 g$* /e/tone 20 g$* 2 Pdetrose glucose3* /E ad!usted to 5.?
Aua dest. sterile $iAc 100 mM sterile $iAc 1 M sterile Bacterial 89A* used as carrier oly7ethyleneglycol >( 50 P w,3 sterile &iltered
10 m$ o& S# 7r/ medium are inoculated with the a//ro/riate yeast strain and incu"ated at 40°C while sha%ing at 200r/m on. -n the net day -# at 00 nm is measured and the yeast culture is diluted with =# to -#00 0.1. Atotal ,olume o& 50 m$ diluted yeast culture is used &or &urther incu"ation. >,ery hour -#00 is measured until-#00 0.6 is reached 4 7 5 hours3. hen the cell num"er is calculated with a homa cham"er. 210< cellsm$are su&&icient &or 10 trans&ormations. he yeast is then har,ested "y centri&ugation at 4000 r/m &or 5 minutes*the su/ernatant is care&ully remo,ed and collected &or autocla,ing. he /ellet is resus/ended in 25 m$ sterile A# and again centri&uged at 4000 r/m &or 5 minutes. A&ter remo,ing o& the su/ernatant the /ellet isresus/ended in 1 m$ $iAc 100 mM. >cess o& $iAc is remo,ed "y s/inning the tu"es &or 15 seconds at &ull s/eedin a ta"leto/ centri&uge and care&ully remo,ing the su/ernatant. he yeast /ellet is "rought to a &inal ,olume o&500 Q$ with $iAc 100 mM. Aliuots o& 50 Q$ are /re/ared. -ne 50 Q$ aliuot o& this yeast sus/ension is used &orone trans&ormation. 50 Q$ aliuots are again "rie&ly centri&uged to /ellet the cells* the su/ernatant is remo,edand on to/ o& the yeast /ellet* layers o& the &ollowing trans&ormation solutions are /i/etted in &ollowing order:260 Q$ 50 P >(* 4 Q$ $iAc 1 M* 4.4 Q$ o& "acterial 89A 41 QgQ$3* <0.< Q$ sterile A#* 1 Qg / lasmid #9A1QgQ$3. he tu"e is then thoroughly mied "y ,orteing &or 1 minute until the yeast /ellet is com/letelydissol,ed and /laced &or 40 minutes in a 40°C water "ath. he tu"e is then trans&erred to a 62°C water "ath &or25 minutes in order to /er&orm the heatshoc%. he trans&ormation mi is then "rie&ly centri&uged &or 15 secondsat 6 000 g < 000 r/m3 in a ta"le to/ centri&uge* the su/ernatant is discarded and the /ellet is resus/ended in1 m$ sterile A#. 50 Q$ o& this trans&ormed yeast sus/ension are /lated on S# D $eu* 7 r/* 7 Ade /lates andincu"ated at 40°C &or some days.
#9A G water G carrier7#9A or 89A 1M $iAc
olyethylenglycol
yeast /ellet
$iAc =east rans&ormation
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136
Mammalian 27Ey"rid System
Limitations:
1. not suited for screeningpurposes
2. Proteins are in the nucleus
and thus eventually not attheir normal localization
Posttranslational modifications such asphosphorylations, which might beessential for interactions are often notcarried out in yeast. In this case asimilar assay can be set up inmammalian cells (e.g. providing thekinase)
> FRET-Microscopy: as alternative tovisualize protein-protein-interaktion in
their physiological context
137
>am/le &or a mammalian 27Ey"rid est
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138
Biochemical eri&ication o& rotein7Interactions
"y Co7Immuno/reci/itation CoI31. The 2 proteins of interest are transfected into mammalian cells (usually containing
to different tags, e.g. HA- and flag). 1 – 2 d after transfection, the cells are lysedand one protein is immunoprecipitated using antibody-beads against tag1 (e.g.flag). The beads are washed extensively with buffer (isotonic or hypertonic, nothypotonic) and finally heated with SDS-buffer to release bound proteins. SDS-PAGE and Western blotting is performed – using antibodies against tag1 andagainst tag2. If protein with tag2 co-precipitated with protein containing tag1, thenthere is interaction.
2. Co-immunoprecipitation of endogenous proteins (without transfection) – using thesame principle and antibodies against the endogenous proteins
Protein
X-myx
Protein
Y-HA1. 2.
bead
Antikörper
3.
HRP
139
>am/le &or a Co7Immuno/reci/itation
1. Co-IP with overexpressed proteins(after transfection); control:transfection with just one protein
2. Co-IP with endogenous proteinscontrol: IP with unrelated antibody
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140
he salt concentration has an in&luence on the
stringency o& the co7immuno/reci/itationCo7Immuno/reci/itation o& 8A1 undIKK2 wor%s at 250 mM 9aCl* "ut not at
500 mM 8A18A2 interaction isstronger than 8A1IKK2 interaction
Co7Immuno/reci/itation o& 8A1 and8A2 at 500 mM 9aCl
141
Co7Immuno/reci/itation &or the #etection o& rotein Interactions
1. Transfection of cells with tagged proteins (one 6-well of CHO or HeLa cells is sufficient for one sample).
2. Preparation of extracts:
2.1. 1 d after transfection: wash cells with PBS
2.2. Lysis with 500 µl/well Lysis-Buffer + Protease Inhibitors: 15 min at 4°C.
Buffer: 0.5% NP40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 150 mM NaCl.
Protease Inhibitors: 10 µg/ml Aprotinin, 20 µg/ml Phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml Leupeptin, 1 µg/ml
Pepstatin (from 1000x stock solutions, Boehringer Protease Inhibitor set).The lysis is suited for cytosolic proteins and
membrane proteins. Nuclei remain intact (you can leave the nuclei on the plate when you take off the supernatant).
2.3. Spin the extracts for 15 – 30 min at 14 krpm, 4°C (HeLas: 15 min, CHO: 30 min)
2.4. Keep the supernatant and adjust the NaCl-concentration (150 mM – 1000 mM depending on the strength of
interaction; start in the range of 150 – 250 mM, increase the concentration if you want to increase the stringency)
3. Co-Immunoprecipitation3.1. Take 400 µl of extract for IP (keep about 30 µl extract for direct western analysis).
Use flat-top tubes (the visibility of the pellet is better in these tubes) Add 400 µl Lysis-Buffer/250 mM NaCl (without
NP40 > final concentration: 0.25%). Add beads (15 µl anti-flag-M2-Agarose, Sigma A-1205; alternatives: other
antibodies directly coupled to CNBr-activated Sepharose; Protein A- or Protein G-Agarose: the later will give more
unspecific binding). Rotate extracts + beads for 2 h at 4°C.
3.2. Spin for 30 sec at 14 krpm 4°C. Take off the supernatant, add 1 ml of lysis buffer/250 mM NaCl/without NP40
and invert tubes several times (do not vortex). Repeat this washing step.
3.3. Suspend the beads in 1 ml cold PBS and transfer the suspension to a new tube.
Spin 30 sec at 14 krpm, 4°C, take off the supernatant and repeat this washing step. Final centrifugation: 1 min at 14
krpm, 4°C. Remove the supernatant and suspend the beads in SDS-PAGE buffer (30 µl). Incubate for 5 min at 95°C
and pellet the beads for 2 min at 14 krpm.
4. SDS-PAGE
5. Western Blot: if possible use HRP-conjugated primary antibodies (anti-HA-HRP from Boehringer, anti-myc-
HRP from Invitrogen). This gives much lower background of unspecific bands (Ig light chain …).
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ar Lestern Blotting
; the mem"rane is /ro"ed with a /rotein* which can "ind the /rotein o&interest. Lhile western "lotting detects certain /roteins using anti"odies* &ar7western "lotting detects /rotein:/rotein interactions.
143
8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions &luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
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144
rinci/le o& luorescence
1. electrons o& a &luoro/hore areecited "y a"sor/tion o& ana//ro/riate /hoton h ν −>3 andtheir energy state is raised to S1´
2. the ecitated state S1´eists &ora"out 1 D 10 nsec. >nergy is lost"y se,eral reactionsinteraction;3 leading to theecited state S1.
4. >lectrons &all "ac% &rom S1 to S0 D the energy di&&erence isreleased "y emission o& a /hoton which has lower energy than theecitation /hoton D and thus a longer wa,elength3 according toλ \ c ν
Ja"lons%i7(ra/h
rinci/les o& luorescence
dou"le "onds \ &lei"le delocalied3 /7electron system
&rom:
htt/:www.in,itrogen.comsiteusenhomesu//ortutorials.html
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delocalied /7electron systems
alternating dou"le "onds3 caneasily a"sor" /hotons andthere"y "e raised to higherenergy le,els
delocalied /7electron systems
alternating dou"le "onds3 caneasily a"sor" /hotons andthere"y "e raised to higherenergy le,els
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energy loss due tomo,ements* rotations etc...
... sudden &all &rom an ecitedenergy le,el to the groundstate
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he a"sor"ance o& light/hotons3 de/ends on thecolour the wa,elength3
num"er o&a"sor"ed/hotons
ecitation s/ectrum
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he emitted light /hotons3ehi"its a certain wa,elengths/ectrum colour3 Dde/ending on the nature o&the &luoro/hore
num"er and colour o&emitted /hotons
emission s/ectrum
153
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154
>citation and >mission S/ectra
Stokes S%ift
In&os: htt/:www./ro"es.comser,letss/ectra
Ja,a7A//let &rom B#: htt/:www."d"iosciences.coms/ectra
155
Ja,a7A//let &rom B#:htt/:www."d"iosciences.coms/ectra
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156
Characteristics o& &luorescent dyes
>citation Maimum: wa,elength o& maimal /hotona"sor"ance λ in nm3
>mission Maimum: wa,elength o& maimal /hotonemission &luorescence* λ in nm3
Molar >tinction coe&&icient: gi,es the a"sor"ance o&ecitation /hotons at the ecitation maimum λ in cm7
1M713
Wuantum7=ield: num"er o& emitted /hotons /er
num"er o& a"sor"ed /hotons.
Brightness \ molar etinction coe&&. uantum yield
157
arameters o& some im/ortant &luorescent dyes
dye > >m
#AI 45@ 61
IC 6@6 520
M8ho 550 5<4
eas8ed 5@5 15
#AI... 4’,6-Diamidino-2-Phenylindol
IC... luorescein Isothiocyanat
M8ho...etramethylrhodamine
8IC: etramethylrhodamine Isothiocyanate3
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158
Alea7luoro/hores &rom Molecular ro"esIn,itrogen
www.in,itrogen.com3
159
( (reen luorescent rotein3 and its ,ariants
• Structure: barrel like with the chromophore in
the middle
• MW: appox. 29 kDa
• original protein from jellyfish (Aequorea
victoria), exists in bacterial and mammalian
codon optimized versions.
• Point mutations were incorporated improvingthe fluorescence (enhanced GFP: EGFP) and
also leading to other spectral variants (colours;
ECFP, EYFP…)
• Fluorescent in living cells – can be expressed
as fusion protein with the protein of interest (is
usually not altering the function of the target
protein)
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160
luorescence ro/erties o& some (7ariants
ariant >citation nm3 >mission nm3
>B Blue3 4?0 660
>C Cyan3 644 6<5
>( (reen3 6?? 50<
>= =ellow3 514 52<
#s8ed 55? 5?4
161
-ther &luorescent /roteins
fluor. protein Ex-Peak nm
Em-Peak nm
quantum yield comment
EBFP 380 440 0.18 Clontech
ECFP 433 (453) 475 (501) 0.4 Clontech
EGFP 488 507 0.6 Clontech
wildtype GFP 397 (475) 509 0.77 Aequorea victoria
EYFP 513 527 0.61 Clontech
Citrine 516 529 0.76 Griesbeck et al. 2001
DsRed 558 583 0.29 Clontech, tetramer
DsRed2 563 582
0.55 tetramer,
HcRed1 588 618 0.02 Clontech, dimer
PA-GFP (Patterson
2002)
400 before act.504 (397)
after
515 before act.517 after act.
0.13 0.79
photoactivatable GFP, T203Hmutant of mammalian
codon-optimized wildtype
GFP
PS-CFP 400 before act.
490 after act.
468 before act.
511 after act.
0.2
0.23
photoswitchable CFP, turns from
cyan to green after intense
illum. at 405 nm
mOrange 548 562 0.69 Shaner et al., 2004
mStrawberry 574 596 0.29 Shaner et al., 2004
mCherry 587 610 0.22 Shaner et al., 2004
dTomato 554 581 0.69 Shaner et al., 2004, dimeric
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hotocon,erti"le &luorescent /roteins
0
20
40
60
80
100
120
0 20 40 60 80 100 120
r e l f l u o r
sec
mOrange conversion to far-red(2x bleaching with 100% 488 nm in between)
mOrange
converted far red
control cell
m-range
ar78ed
hoto7switcha"le &luor. /rotein #ron/a
0
20
40
60
80
100
120
-20 0 20 40 60 80 100 120 140 160 180 200
% o
f i
n i t i a l f l u o r
sec
start bleaching at 488 nm
start reactivation
at 350 nm
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164
luorimetric Analysis Methods
luorescence measurements are usually more sensiti,e than/hotometric measurements.Scanning &luorometers ha,e usually 2 monochromators &or ad!ustingecitation and emission wa,elengths. Most instruments also allow toad!ust the "andwidth o& ecitation and emission "etween 1D20 nm3he emitted &luorescence is measured "y a /hotomulti/lier tu"eM3 detector. he sensiti,ity o& that can "e ad!usted "y changingthe ,oltage e.g. 600 7 <00 3.
>mission
Monochromators#etector
165
arameters o& &luorometry
>citation wa,elength in nm
"andwidth o& the ecitation light 1 D 20 nm* Hslit width3
>mission wa,elength in nm
"andwidth o& the emission
sensiti,ity o& the detector Hgain* ,oltage o& the M3
Integration time o& the measurement slow D &ast* in sec.:in&luences the Hnoise3
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166
La,elength Scans
0
0.5
1
1.5
2
460 480 500 520 540 560 580
nm
r e l . f l u o r .
>mission s/ectrum >C3
he eact emission andecitation /ea%s might di&&erslightly "etween di&&erent&luorometers.
or chec%ing the /arameters:7 run an ecitation wa,elengthscan at the literature ,alue o& the/ea% emission7 run an emission scan at thedetermined ecitation /ea% 7 re/eat the ecitation scan at
the determined emission /ea% or ad!usting these /arametersyou ha,e to consider the&luorescence /ro/erties e.g. theSto%eOs shi&t3 D to /re,ent thatecitation light is detected
ric%s &or o/timiing &luorescencemeasurements "ased on the s/ectra
nm
luor.
theor.ecitationcur,e
theor.emissioncur,e
real
emissioncur,e 1
ecitationwindow
/ossi"le emissionwindow
detected &luorescence
s/ill7o,er o& the ecitation light
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ric%s &or o/timiing &luorescence
measurements "ased on the s/ectra
nm
luor.
theor.ecitationcur,e
ecitationwindowmorenarrow
emission cur,e 2
detected &luorescence
emission window
ric%s &or o/timiing &luorescencemeasurements "ased on the s/ectra
nm
luor.
real
emissioncur,e 1
real emission cur,e 4
le&t shi&ted"roader ecitation
window
detected &luorescence
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170
ime Scans
0.001
0.003
0.005
0.007
0.009
0.011
0.013
0.015
0.017
0 5 1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
1 0 0
1 0 5
1 1 0
min
E
x 4 3 6 / E m
5 1 0
-0.01
0.01
0.03
0.05
0.07
0.09
0.11
0.13
0.15
0.17
0.19
T r p - f l u
o r e s c e n c e
SERT
Standards
670 kDa
158 kDa
44 kDaSERT
Can "e a//lied to determine the time course o& &luorescence changes e.g. todetermine enyme reaction %inetics D or &or instance in chromatogra/hy tomeasure the %inetics o& elution and thus the molecular weight o& a &luorescentcom/ound* such as a (7&usion /rotein3
171
ast Kinetic7AnalysisSto//ed7low luorometry3
wo reaction /artners are in!ected into a miing cham"er* where they are miedwithin a//ro. 1 msec "y sto//ing the &low.I& the reaction "etween the two com/ounds changes the &luorescence* this changecan "e recorded with a resolution in the microsecond range.
A
B
>citation light
luorescenceSto//ed low luorometry3
$ight7A"sor"anceSto//ed low hotometry3
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172
>am/le &or a sto//ed7&low &luorometry
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0 0.02 0.04 0.06
seconds
r e l . f l u o r e s c e n c e
Tet-DNA
mutant T et-DNA
y = 734,21x + 244,09
R2 = 0,9508
0
100
200
300
400
500
600
700
800
900
0 0,2 0,4 0,6 0,8
DNA (µM)
r a t e ( 1 / s e c )
rate (15°C)
rate (37°C)
angent o& the initial &luorescence change reaction %inetics rate in 1sec3
$inear correlation "etween the initialreaction %inetics range and theconcentration o& the reaction/artners
K a&& \ 1K diss \ % on % o&&
% o&&
% on
% o&& % on
173
Wuantitati,e luorometry
1. >nyme7Measurements e.g. β7(alactosidase3
2. #9A7Measurements Eoechst 4425?* S=B8 (reen3: &luorescentdyes* which intercalate into the #9A and are &luorescentde/endent on the amount o& #9A
4. rotein7Measurements inherent &luorescence due to aromaticamino acids such as ry/to/hane dye: S=B8 -range* ;3
luorescence measurements can "e used to uanti&y a great ,ariety o&di&&erent su"stances.+sually a standard cur,e is measured with the o/timied measurement/arameters e.g. a&ter de&ining them "y wa,elength scans: ecitationand emission wa,elengths and corres/onding "andwidthsF M ,oltageand integration time3.
Some eam/les &or uantitati,e &luorometry
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174
>am/le &or uantitati,e &luorimetricmeasurement
y = 0.8738x + 0.1417
R2 = 0.9959
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8
µg/ml
F l u o r e s c e n c e
( E m
3 7 6 / E x
2 7 6 ) ro+e
Standard cur,e
175
Con,entional C8:
Com/arison o& a gene o& interest with a house%ee/inggene using an end/ointdetermination
Sam/le 1 2
In real: sam/les contain di&&erentamounts o& c#9A* "ut this di&&erenceis not detected* when they reach thesame /lateau at the end o& thereaction this can "e re,ealed "ymeasuring the reaction /roduct "y&luorescence3 a&ter each cycle
1
2end/oint
luorescence as measurement ,alue in s/ecialanalysis techniues: 8eal7ime C8
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176
C8 rinci/le
177
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178
179
$ightCyclerM* 8oche
ca/illary withthe C87mi
heating
entilator
light source
dichroic mirrors
detectors
threshold
Ct
Scheme o& a C8 machine
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8ealtime C8 machines
180
A//lied Biosystems
Ste/-ne lus8oche $ight Cycler
ca/illaries
a//. 1 Rsam/le3
@7well /lates
181
8eal7ime C8 with S=B8 (reen as #9A7&luorescence dye
S=B8 (reen intercalates in theam/li&ied ds#9A C87/roduct37leading to an increase in &luorescencewith increasing cycle num"er.
A&ter many cycles the &luorescencealso increases in the water control Ddue to the &ormation o& /rimer
aggregates
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182
Melting /oint analysis o& the C87/roduct
S/eci&ic and uns/eci&ic C8/roducts can "e distinguished"y their di&&erent meltingtem/erature as determineda&ter the C8 "y slow heatingand the decrease o& the&luorescence at the melting/oint3. his can also "e a//liedto detect mutations.
measurement o& the &luor. a&ter each cycle at atem/erature a"o,e the melting tem/erature o& theuns/eci&ic C8 /roduct allows uanti&ying !ust thes/eci&ic /roduct
183
8eal7ime C8 with 8>7Ey"ridisation ro"es
Lithin the seuence &lan%ed "y rimer 1 andrimer 2 am/li&ication /rimers3* two additionaloligonucleotides Ey"ridisation ro"es 1 and 23are situated* which contain two di&&erent&luoro/hores at the 4O and 5O ends. Lhen theseoligos "ind to the C8 /roduct* the &luoro/horescome into close /roimity and one &luoro/horecan trans&er /art o& its &luorescence energy to theother one &luorescence resonance energytrans&er* 8>3* which then starts to shine. In
this case /rimer aggregates do not generate a&luorescence signal.
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184
8eal7ime C8 with aMan /ro"es
A aMan7/ro"e contains 8>7#onor and Acce/tor Wuencher37luoro/hore within thesame oligonucleotide. At the annealing tem/erature o& the oligo* the /ro"e "inds to the C8/roduct. he eonuclease acti,ity o& the a7olymerase clea,es the /ro"e and leads toincrease o& the #onor7&luorescence due to de7uenching.
8ealtime C8 D Wuanti&ication o& geneu/down7regulation
y = -1.67ln(x) + 25.767R² = 0.9972
15
17
1921
23
25
27
29
31
33
35
0.1 1 10 100
C P
ng cDNA input
PCR efficiency
crossing point CP
Log. (crossing pointCP)
1. #etermine the C8 e&&iciency o& yourgene o& interest and that o& yourhouse%ee/ing re&erence3 gene usingserial dilutions e.g. o& /lasmids orc#9A /re/arations3: > \ ideally 2du/lication at each cycle3 "ut
realistically lower e.g. 1.?3
2. Calculate u/7 or downregulation o& your s/eci&ic gene o& interest usingthe di&&erences in the crossing /ointC3 ,alues with the euation:
>cel tem/late on my we"site3)(
)(
arg
)(
)( arg
samplecontrolCP
ref
samplecontrolCP
et t
ref
et t
E
E ratio
−∆
−∆
=
Ct7method
&a&&l ML: A new mathematical model &orrelati,e uanti&ication in real7time 87C8.9ucl Acids 8es 2001* 2@@3:e65
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Calculating the C8
e&&iciency &rom thesha/e o& the cur,e
186
10
100
1000
10000
100000
0 10 20 40 60 50
l o g &
0 l u o r (
cycle
710000
0
10000
20000
40000
60000
50000
0000
<0000
?0000
0 10 20 40 60 50
0 l u o r
cycle
7 Lithout using a dilution cur,e
7 Can "e calculated &or eachsam/le se/arately
>cel tem/late on my we"site3
9eurosci $ett. 2004 Mar 14F44@13:27.
Assum/tion7&ree analysis o& uantitati,ereal7time /olymerase chain reaction C83data.
8ama%ers C* 8ui!ter JM* #e/re 8E*Moorman A.
$in8egC8 So&tware
187
htt/:www.hart&aalcentrum.nlinde./h/^main\&iles`su"\$in8egC8
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188
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8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions &luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
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Microsco/y: Euman ,ision and the conce/t o&
magni&ication
190
image &ormation in the human eye
27ste/ magni&ication /rinci/le o& amicrosco/e with 2 lenses:
o"!ecti,e and eye /iece occular3
191
Basics o& o/tical resolution I
ine structures induce a di&&raction o& light lighto& ero7order* 1st order ...3. $ight di&&raction on asmall iris is more or less eual to di&&raction onsmall cellular structures:
sinθ
(1)
1.22(λ
/d)
θ ... angle to the &irst light minimumλ... wa,elengthd ... diameter o& the iris
for very small angles θ: θ
(1)
1.22(λ
/d)o"!ects that are closer than
θ
(1) cannot "e resol,ed asse/arate o"!ects
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Basics o& o/tical resolution II
he more orders o& light are resol,ed the "etter is the resolution.
he o/tical resolution that can "e achie,ed is de&ined "y the so callednumerical A/erture 9.A.3 o& the o"!ecti,e.
#.A. B i sin @
i ... 8e&raction inde o& the mediume.g. 1.0 &or air* u/ to 1.5 &or oil3
... hal& o& the o"!ecti,e o/ening angleA/erture3
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HAiry dis%s: o/tical "asic structures
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#eceleration /hase shi&t3 o& light "y /assingthrough an o"!ect
htt/:www.microsco/yu.comtutorials!a,a/hasecontrast/hases/ecimensinde.html
195
Khler Illumination
; was esta"lished to guarantee o/timalillumination o& o"!ects.
his illumination is usually also a/rereuisite &or di&&erent contrast methods/hase contrast* di&&erential inter&erencecontrast3 to wor%* as the necessary
com/onents are o/timied &or Khlerillumination.
In order to get a Khler illumination* youha,e to &ocus the o"!ect &irst* then youclose the &ield iris* so that !ust the middle/art o& the ,iew &ield is illuminated i&necessary you ha,e to center the light/ath3 and then the ,ertical /osition o& thecondensor is ad!usted so that the "orderso& the &ield iris a//ear shar/ and &ocussed.
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196
Correct and wrong illumination
Correct Koehler illumination
&ield iris at wrong ,ertical /osition o& the condensor
light /ath not centered
197
S/eci&ications o& o"!ecti,es
Magni&ication 10* 20* 60;3: totalmagni&ication is gi,en "y o"!ecti,emagni&ication and occular magni&ication ormagni&ication o& the lens in &ront o& the CC#camera3
Immersion medium: air* oil D or waterEigh magni&ication 60 D 1003 with oil orwater
additional &eatures e.g. suita"ility &or&luorescence due to low auto&luorescence o&the glass: 9eo&luar..3
contrast &eatures: e.g. /hase contrast e.g.h13
correction o& lenses &or chromatica""erations e.g. A/ochromat3
correction o& lenses &or /lanarity o& &ocuso,er the ,iew &ield lan3
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Contrast enhancement in transmitted light
microsco/y
4. #i&&erential7Inter&erence contrast9ormars%i3: Ma%ing use o& light/olariation and its change througho"!ects to generate a contrast
2. hase contrast:Ma%ing use o& the/hase o& light when
it /asses an o"!ect
1. Staining o& structurese.g. nucleus: "lue withhematoylin* antigen:"rown with immuno7histochemistry3
199
hase contrast
+nstained o"!ects such as cellsslow down the light the /hase o&the /assing light3 "y λ. hasecontrast rings in the o"!ecti,e canaccelerate the light* which doesnot /ass through cells "y λ* theresulting di&&erence o& f λ causesan inter&erence* which leads to
contrast enhancement. λ
f λ
htt/:www.microsco/yu.comtutorials!a,a/hasecontrasto/ticaltraininde.html
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hase contrast II
he /hase contrast rings o& the o"!e%ti,e and the condensor ha,e to match each other indiameter and ha,e to "e concentric. In addition the distance "etween them has to "ecorrect which is the case at the Khler illumination3 D this is es/ecially im/ortant &orhigher magni&ication o"!ecti,es. It is stated on the o"!e%ti,e which /hase contrast ring hasto /ut in at the condensor e.g. h1* h2...3.
201
/hase contrast III
wrong /hase contrast ring
/hase contrast rings not centered
correct Koehler illumination and/hase contrast
htt/:www.microsco/yu.comtutorials!a,a/hasecontrastmicrosco/ealignmentinde.html
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Illumination scheme o& an in,erted microsco/e
condensor with /hasecontrast rings
Kno" to ad!ust the,ertical /osition o& the
condensor
&ield iris
screws &or centering thelight /ath
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#i&&erential7Inter&erence7Contrast #IC39ormas%i Contrast3
Be&ore reaching the condensor* the light is /olaried and/asses a dou"le /rism Lollaston rism3* where it is s/litinto two "eams with di&&erent directions and/er/endicular wa,es. hese "eams /ass the sam/le*where they are altered in intensity and /hase etc. he"eams are &ocussed "y the o"!ecti,e. In the &ocal /lanethere is a second dou"le /rism* which com"ines the
"eams again. A&ter that* the "eams are de/olaried again.here"y the "eams that ha,e "een altered di&&erentially inthe sam/le can inter&ere with each other D and thisinter&erence results in changes o& the intensity and thecolour.
he outcome is a /reudo7threedimensional image.
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Com/arison
hase Contrast D #i&&erential Inter&erence
Ee$a cells D same ,iew &ield
htt/:www.microsco/yu.comtutorials!a,a/hasedicmor/hinde.html
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luorescence Microsco/y
>am/le: ri/le7luorescence7la"eled
endothelial cells:8ed: Actin7ilamentsla"eled with halloidin
(reen: Mem"ranes #i-7C3
Blue: 9uclei #AI7stainingo& #9A3
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Basics o& &luorescence microsco/y
luorescent sam/les areecited with light o& ana//ro/riate wa,elengththrough the o"!ecti,e3* theemitted &luorescence iscollected again "y theo"!ecti,e and is guided to adichroic mirror* whichse/arates the ecitation light&rom the emitted&luorescenceF the latter
/asses an emission &ilter andis detected "y eye or "ya//ro/riate detectors such ascameras3
Interacti,e )eiss7utorials: htt/:eiss7cam/us.magnet.&su.edututorials
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Scheme o& a &luorescence microsco/e
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$ight sources &or &luorescence ecitation
1. Con,entional light sources:
7 mercury lam/s:
7 Yenon7lam/s:
$># $ight Sourceslight7emitting diodes: semiconductor de,ices3
&rom: htt/:eiss7cam/us.magnet.&su.edu
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Metal Ealide $am/s
&rom: htt/:eiss7cam/us.magnet.&su.edu
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$aser light sources
2. $aser $ight Am/li&ication "yStimulated >mission o& 8adiation3:
(i,e !ust discrete wa,elengthslines3 D thus the choice o&ecitation light is limited andde/ends on the laser ty/e.
Ar7laser: main lines at 6?? nm and
516 nm* and 65?3Ee9eon: 564 nm* 44 nm
+7$aser: 605 nm
iolet laser diodes: 605 D 620 nm
Ad,antages o& laser light:
7high uality /arallel light "eams37good &or scanning
7high intensity
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luorescence ilter Cu"es
he &ilter cu"e consists o&:
1. >citation &ilter: !ust thecorrect ecitation lightwa,elength3 /asses the &ilter
2. #ichroic mirror: is re&lecti,e&or the ecitation light "uttransmittent &or the emissionlight the emitted &luorescence3
D se/arates ecitation &rom&luorescence light
4. >mission &ilter: &ilters theemitted light so that !ust thecorrect wa,elength e.g. indou"le &luorescence3 reachesthe detector
sample
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Characteristics o& &luorescence &ilter sets
>citation &ilterBand ass3
>mission &ilterBand ass3
emission &ilter
ecitation &ilter
dichroicmirror
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>am/le o& a "and/ass &ilter G nomenclature
214
215
#ual"and &ilter sets: Simultaneous o"ser,ationo& two di&&erent &luoro/hores e.g. >(#s8ed3
>citationdichroic mirror
>mission
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Monochromators as light source
A con,entional light source e.g. a Yenon lam/3 is s/lit "y amonochromator e.g. a di&&raction grid3 to the s/ectral colours Dto /roduce light o& a &reely de&ina"le wa,elength 420 D <00 nm3.his can "e used instead o& a &ied ecitation &ilter.
-ne ad,antage is that this technology allows switching "etweendi&&erent ecitation wa,elengths within &ew milliseconds. his can"e im/ortant &or ecitation ratio imaging e.g. ura72 Calciumimaging etc.3
+ 420 nm3
8ed <00 nm3electronicallyad!usta"le grid
olychrome &romI$$ hotonics
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#etection o& the emitted &luorescence7 isually ,ia the occular o& the microsco/e
7 "y a CC# camera usually cooled to reduce the electronic noise3.he /hotons o& the &luorescence hit a light sensiti,e chi/ e.g. outo& 1400 1040 /iels3* where electrons are released de/endenton the intensity o& the &luorescence. >ach chi/ can resol,e a gi,enintensity range D e.g. 25 grey ,alues &or a ?7"it camera or 6000grey ,alues &or a 17"it camera.
he images can "e shown on a com/uter monitor and sa,ed on acom/uter
7 "y /hotomulti/lier tu"es MOs3: used o&ten &or scanningde,ices such as con&ocal laser scanning microsco/es. he gain,oltage3 o& the M de&ines the sensiti,ity electrons released &ora gi,en num"er o& /hotons that hit the detector3. -&ten more
Hnoisy than CC# camera images. A,eraging is used to smooththe images good images ta%es a"out 6 sec D while CC# reuire
!ust a"out 100 msec3.
7 old &ashioned: &ilm camera and sensiti,e &ilm e.g. 100 ASA3
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>am/le &or a &luorescence microsco/y
e/eriment7 Cells trans&ected with &luorescent &usion /roteins o& a transcri/tion&actor and its inhi"itor a//ear in the cytosol3F
7 addition o& le/tomycin B $MB3 to "loc% nuclear e/ort. his leads toaccumulation in the nucleus indicating continuous nucleo7cyto/lasmicshuttling
219
>am/le &or a &luorescence microsco/ye/eriment II
0 20 40 60 800
1
2
3
4
5
min
c y t o s o l i c / n u c l e a r f l u o r
.
Fluorescence was quantified in the nucleus and in the cytosol of the same cellafter different time points > shows the kinetics of nuclear accumulation by thechange of the cytosolic/nuclear ratio.
The data were fitted by nonlinear regression analysis (single exp. decay) –
leading to the half time of the nuclear import process.
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rotocol o& an immuno&luorescence staining
iation: 15 min 6P ara&ormaldehyd
4 5 min mit BS wash 50mM ris7ECl /E<.6* 150 mM 9aCl* 0.1Priton3
Bloc%: 1 h at 8 with 4P BSA in BS
Incu"ation with 1. A": anti7Iκ B ra""it/olyclonal* sc74<1 Santa Cru31:400 in BS4P BSA* o,er night at6NC or 1 h at 4<NC3.
2 5 min wash with BS* 1 5 minwith BS
Incu"ation with Alea6?? goat anti7ra""it 1:2000 in BSBSA: 1 h at 4<NC
4 5 min wash with BS* 1 5 minwith BS
Mounting
221
Com"inations o& transmitted light and &luorescence
A3 directacuisition with"oth light sources
B3 Se/arate acuisition o& &luorescence and /hase contrast and mergeor "lending e.g. with ImageJ or other so&tware3
I7$a" So&tware
ImageJ so&tware
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222
Interacti,e Microsco/y #emonstrations
9ery recommenda+le:
%ttp:micro.magnet.fsu.edu
1. Optical resolution:htt/:www.microsco/yu.comtutorials!a,aimage&ormationairynainde.htmlhtt/:www.microsco/yu.comtutorials!a,alightandcolorre&ractioninde.html
2. <D%ler 'llumination:htt/:www.microsco/yu.comtutorials!a,a%ohlerinde.html
5. %ase s%ift of lig%t +y an o+ecthtt/:www.microsco/yu.comtutorials!a,a/hasecontrast/hases/ecimensinde.html
6. %ase contrasthtt/:www.microsco/yu.comtutorials!a,a/hasecontrasto/ticaltraininde.html
htt/:www.microsco/yu.comtutorials!a,a/hasecontrastmicrosco/ealignmentinde.html
. O+ecti)es ,it% adusta+le ,orking distancehtt/:www.microsco/yu.comtutorials!a,aa"errationscorrectioncollarinde.html
223
Con&ocal $aser Scanning Microsco/y C$SM3
hotomulti/lier
con&ocal /inhole
dichroic mirror
-"!ecti,e
7Motor
Scanner
$aser
ro"lem in con,entional microsco/y: light* which comes &rom outside o& the &ocal /lanea"o,e or "elow3 gets to the detector or eye3 and is registered as "lur* which decreases theuality o& the image
Solution: A /inhole iris3 is /laced into the light /ath at a /osition* where it can "loc% out7o&7&ocus light. By that means an o/tical section is imaged with ,aria"le thic%ness starting witha//ro. 0.? Qm3 de/ending on the diameter o& the /inhole.
+sually high uality ecitation light is needed &or that e.g. coherent laser light with /arallellight "eams3. he result is a ,ery shar/ image without any "lur &rom out7o&7&ocus light with a
slightly higher resolution than with con,entional e/i&luorescence microsco/y.
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Con&ocal microsco/y remo,es the "lur&rom thic%er o"!ects
htt/:eiss7cam/us.magnet.&su.edututorialso/ticalsectioningcon&ocalwide&ieldinde.html
-/tical sectioning and 4#7/ro!ections
225
Acuisition o& a H7stac% image slices along the 7ais3 allows reconstruction o& a 4#7/ro!ection* whichcan "e shown as animation
7stac%
4# rendering/ro!ection
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S/ectral imaging
H>mission &inger /rinting: emission scan o& amicrosco/y sam/le Hlam"da stac% o& images3 at agi,en ecitation wa,elength e.g. with )eiss $SMM>A systems or with $eica con&ocal microsco/es;3
Alternati,e: >citation scan at a constant emissionwa,elengthF e.g. using a monochromator lightsource3
Com"inations o& ecitation and emission &inger/rinting e.g using &ilter wheels3
Increases the num"er o& mar%ers to "e measured in
/arallel Can "e used to discriminate &luoro/hores with
o,erla//ing s/ectra
Can "e used to discriminate s/eci&ic &luorescence&rom auto&luorescence
8esol,ing s/ectral in&ormation on a /iel7"y7/iel "asis
$eica conce/t
)eiss M>A conce/t
227
lambda7stac%
Spectral curve of aregion of interest
$eica Con&ocal Microsco/e CS S2: Monochromator in &ront o& the detector A-BS: Acousto7-/tical Beam S/litter instead o& dicroic mirror3
M>A System o& )eiss: 42 M7detectors e,ery 10.< nm600 D <20 nm3: simultaneous wa,elength analysis.
S/ectral Imaging Con&ocal Microsco/ywith >mission Cur,e Analysis3
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Zimmermann et al.
(FEBS Letters 2003) Sample with overlappingfluorophores
Emission curves separated
into 8 channels (left) or
2 channels (right)
Equation matrix for the
channel signals based on
reference intensities in the
channels (GFPn and YFPn)
and the unknowncontributions of the
fluorophores
Unmixed fluorescence
(pseudo-coloured)
1 2 4
6 5
< ?
1
2
S/ectral imaging eam/le I: C* ( and =
229
htt/:eiss7cam/us.magnet.&su.eduarticless/ectralimagingintroduction.html
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S/ectral imaging eam/le II: strongly
o,erla//ing dyes
230
S=-Y (reen nucleus3* Alea luor6?? con!ugated to /halloidin&ilamentous actin networ%3* and-regon (reen 516 con!ugated to goatanti7mouse /rimary anti"odiestargeting mitochondria3.
htt/:www.in,itrogen.comsiteusenhomeroducts7and7Ser,icesA//licationsCell7
Analysis$a"eling7Chemistryluorescence7S/ectraiewer.html
In,itrogen S/ectra iewer
Se/aration o& s/eci&ic &luorescence &rom auto7&luorescence "y s/ectral imaging
231
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>am/le &or >mission inger/rinting on a )eiss
$SM510 M>A: Se/aration o& ( and =
Acquisition of a
reference lambda
stack for the first
fluorophore (GFP)
500 510 520 530 540 550 560Emission wavelength (nm)
0
50
100
150
200
250
Intensity
Obtain the spectral emission curve for the first fluorophore and
repeat the procedure for the second fluorophore
GFP
500 510 520 530 540 550 560Emission wavelength (nm)
0
50
100
150
200
250
Intensity
YFP
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+nmiing o& a mied sam/le
(7Actin and =7mem"ranes3
Emission stack
Unmixed image
235
image with mied signals &ordi&&erent mar%ers
+nmiing o& signals in /athology sam/les
auto&luorescence
Bright&ield dis/lay
Shown with the 9uance7So&tware &rom Cam"ridge 8esearch ` Instrumentation3
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H8ealtime con&ocal microsco/y* S/inning dis%
con&ocal microsco/y with 9i/%ow7dis%s3
gentle scanning less "leaching good &or sensiti,e li&e cells
#etection o& the signal with a CC#7camera
htt/:eiss7cam/us.magnet.&su.edututorialss/inningdis%yo%ogawainde.html
237
Com/anies &or con&ocal microsco/es
)eiss: htt/:www.eiss.de
$eica: htt/:www.leica.comwww.confocal-microscopy.com
• Nikon: http://www.instrumente.nikon.de/
• Olympus: http://www.olympus.de/microscopy/
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Multi/hoton $aser Scanning7Microsco/y
A uantum /hysical /henomenon is used: at ,ery high lightdensities using /ulsed lasers* a"out @00 nm in&rared light3/ac%ages o& 2 or more /hotons occur !ust in the &ocal /lane U3.hese ha,e the same energy as single /hotons o& higher energyshoerter wa,elength* e.g. 650 nm3. hus these /hoton /ac%agescan ecite a &luoro/hore* which emits then at &or instance 520 nmmitted wa,elength is horter than the ecitation light wa,elength U3.
An im/ortant ad,antage is that the @00 nm light has a mucherdee/er /enetration into tissue a//ro. 1 mm3* while con,entionalecitation can image !ust down to 0.25 mm. Another ad,antage is a
reduced o,erall "leaching e&&ect* as ecitation /hoton /ac%agesoccur !ust in the &ocal /lane.
239
Multi/hoton $aser Scanning7Mi%ros%o/ie II
con,entional ecitation17hoton cone o&ecitation light3
27hoton ecitation:only a s/ot o&
ecitation
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S/ecial luorescence Microsco/y echniues
1. 0!A: luorescence 8eco,ery A&ter hoto"leaching
2. 0$': luorescence $oss in hoto"leaching
5. 0!ET: luorescence 8esonance >nergy rans&er
6. 0$'4: luorescence $i&etime Imaging Microsco/y
. 0'S8: luorescence In Situ Ey"ridiation
241
8A: luorescence 8eco,ery A&ter hoto"leaching
An image is ta%en D then a region o& the cell is "leached "y high laser intensity* &ollowed "ya time series o& images a&ter "leaching. Brie&ly a&ter "leaching the region is signi&icantlydar%er and then the &luorescence intensity increases again &luorescence reoo,ery3 due todi&&usion o& molecules into the "leached area. he %inetics o& reco,ery de/ends on thedi&&usion coe&&icienceF the etent o& reco,ery the /lateau to which the &luorescencereco,ers3 is a measure o& the o,erall mo"ility the &raction o& mo"ile molecules ,ersusmolecules immo"ilied* e.g. to the cytos%eleton3
8A in the cytosol:
8A at the mem"rane
Non linear regression analysis
y = span (1-e-kx) + bottom
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in,erse 8A with no,el &luorescent/roteins
243
rotocol: 8A analysis on )eiss $SM510 Ca/ture an image o& the whole cell "e&ore "leaching #e&ine a "leaching scan region and may"e in addition another scan region that is not
"leached3 er&orm a time series with 1 scan /re"leach* a"out <0 iterations o& "leaching with 100P
laser /ower and then 507100 scans o& the "leach region and also the non7"leached controlregion i& you s/eci&ied one37 a good time resolution can only "e o"tained i& !ust the small"leach region and may"e the control region3 is scanned 7 and not the whole cellFa,eraging o& 2 or 6 scans reduces the electronic noise and leads to "etter uanti&ications.
Ca/ture an image o& the whole cell a&ter the 8A time series with the same conditions asthe /re"leach image D &or calculating the total loss o& &luorescence.
I& you want to sa,e dis% s/ace: etract the 8A region and sa,e !ust this region instead o&
the whole image It is recommended to use the LCI ,ersion o& ImageJ &or analysis: =ou can o/en the $SM7
&iles with the "uilt7in &eature which also allows o/ening the time ,alues o& the imageseries3. Measure the mean &luorescence in a control region or &or the whole cell &or "oth the/re"leach and the /ost"leach images and calculate the loss o& o,erall &luorescence due tothe "leaching in the region o& interest this is necessary &or o"taining correct reco,ery,alues &or the "leach region3.
Im/ort the 8A7image seuence* de&ine a measurement region and a//ly the Hintensity,ersus time /lot /lug7in D this will draw a gra/h o& the 8A cur,eF clic%ing the list "utton*shows a list o& the numerical ,alues the &irst 6 /arameters are dimension and /osition o&the region* the rest are the &luorescence intensity ,alues3.
Co/y the &luorescence raw data &rom the list to the corres/onding column o& an >celtem/late
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bottomespan y kx
+−×= −
)1(
Calculate the di&&erence o& mean &luorescence &rom the "ac%ground and normaliethe &luorescence ,alues to 100P &or the initial &luorescence.
#i,ide the /ercent ,alues "y the correction &actor calculated &rom the total loss o&&luorescence e.g. i& total &luorescence decreased &rom 1 to 0.@ then di,ide themean &luorescence o& the 8A regions &or each time ,alue "y 0.@ to com/ensate&or the loss in total &luorescence3. A similar com/ensation can "e o"tained "ynormaliing the 8A &luorescence ,alues to the control scan region that was not"leached. his method also com/ensates more eactly &or the "leaching e&&ect inthe course o& scanning o& the time series this scanning7de/endent "leachinge&&ect is o//osed to the reco,ery o& &luorescence in the "leach region due todi&&usion o& non7"leached molecules in the "leach region3. his dynamiccorrectionX gi,es a somewhat "etter estimation o& the cur,e and the %inetics o&the reco,ery3 D "ut leads in /rinci/le to results that are ,ery similar to the cur,eo"tained with the constant correction &actorX "y calculating the total loss in&luorescence "ased on the intensities o& the images that were ca/tured "e&ore anda&ter the 8A7time series3
or non7linear regression analysis cur,e &it o& the data to a single e/onentialassociation algorithm3: Co/y the data to a &itting /rogram such as (ra/hadrism3 and /er&orm the &itting with a "ottom to s/anX algorithm:
$I: luorescence $oss in hoto"leaching
; to determine the dynamic shuttling o& molecules "etween di&&erentcom/artments o& the cell
A certain com/artment A e.g. the cytosol3is re/etiti,ely "leached "y the laser D andthe &luorescence decrease in a di&&erentcom/artment B is monitored "y time la/semicrosco/y. Molecules that shuttle &rom Bto A are "leached in A thus thecom/artment B gets dimmer when there isa dynamic distri"ution o& molecules"etween A and B. 0
20
60
0
?0
100
120
0 2 6
cytosol
nucleus
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$I to determine a nuclear e/ort signal and anucleolar localiation signal
nuclear FLIP(bleach in nucleus outside nucleoli)
0 100 200 300 400 500 600 7000
25
50
75
100
125nuclear
nucleolar
sec
9B inducing %inase
truncated 9IK without the e/ort seuence:
CS: luorescence Correlation S/ectrosco/y
; to determine di&&usion coe&&icients and interactions "etween molecules.he sam/le is illuminated "y the laser at a ,ery small s/ot* the mo,ementso& molecules in this s/ot in and out3 cause &luorescence &luctuations* whichare analyed "y correlation &unctions
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8>: luorescence 8esonance >nergy rans&er
Microsco/y>nergy can "e trans&erred "etween two &luoro/hores when theyare ,ery close to each other closer than 10 nm3 and when theemission cur,e o& one the energy donor3 o,erla/s with theecitation cur,e o& the other one the acce/tor3. his trans&er o&energy does not ha//en ,ia /hotons U3 "ut "y a di/ole7interactiona uantum /hysical /henomenon disco,ered "y heodor 0Drsterin 1@63. As a result the donor &luoro/hore &luorescence "ecomeswea%er and the acce/tor &luorescence increases.
he 8> e&&ect decreases with the th /ower o& the distanceF thedistance o& hal& maimal energy trans&er is called drster 7#istance8
0
&or C and = it is a//roimately 5 nm3. As the e&&ect isusually not detecta"le anymore at a distance higher than 10 nm itis ideally suited &or monitoring macromolecular interactions/rotein7/rotein or /rotein7#9A3. By that means* not only theinteraction "y itsel& can "e detected* "ut also the location o& theinteraction and its dynamics.
249
rinci/le o& luorescence 8esonance >nergy rans&er
excitation
emission
nm
Donor Acceptor
> \ 8 0 8 0
G r3 und
8 0 \ κ 2 × Jλ3 × n76 × WT1 × @<0
80 ; rster7#istancer ;.. real distanceκ ;.. -rientation &actorJλ3 ; s/ectral o,erla/n ; re&racti,e indeW ; Wuantum yield o& &luor.
DonorAcceptor
ecitation
no FRET FRET
donor fluor.
(CFP)
acceptor fluor.
(YFP)
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A//ro/riate luoro/hore airs &or 8>
luoro/hore air Comments
C0 F0 (ood com"ination &or normal 8> microsco/y using Eg7lam/s as light source and s/ecial &ilters. C is /oorlyecitated "y Ar7lasers* "ut: good ecitation "y "lue laserdiodes
G0 =0 B has in&erior &luorescence /ro/erties
=0 "s!ed-)ariants -riginal #s8ed is !ust &luorescent as tetramer* showscom/le maturation characteristics with green &luorescentintermediates and tends to aggregateF #s8ed2 is a dimer.
he new monomeric #s8ed wor%s ,ery &ine with (.
=0 F0 Are ,ery di&&icult to se/arate with &ilters "ut can "e used in$IM and with s/ectral analysis3
=0 Cy5 or Alexa 6 Anti"odies can "e directly la"eled with Cy4 or eui,alent Alea dye and gi,e 8> with a ( chimera to which they"ind.
0'TC T!'TC Classical 8> /air &or la"eled /roteins e.g. anti"odies3
Alexa 633 Alexa 6
&Cy5 Cy(
alternati,es as la"eling dyes su/erior to IC and 8IC3
251Tet-la+el
=0(797κ B
>citation o&( 6?? nm3
8>
(7>mission 512 nm3
et7>mission 560 nm3
#9A
8> can "e used to monitor /rotein7#9Ainteractions
0.0
0.2
0.4
0.6
0.8
1.0
1.2
480 500 520 540 560 580 600
nm
r e l a t i v
e f l u o r e s c e n c e
GFP-NFkB (Em)
Tet (Em)
Tet (Ex)
Spectral overlap
S/e%tral analysis o& a miture: Increase inacce/tor &luorescence indicates 8>
s/ectra o& donor and acce/tor (797κ Band et7la"eled #9A* res/ecti,ely3
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X Y
Donor Acceptor
FRET
1 nm
ECFP and EYFP-Scans
0,0
0,2
0,4
0,6
0,8
1,0
1,2
350 400 450 500 550 600
nm
r e l a t i v e f
l u o r e s c e n c e
EYFP (Em)
EYFP (Ex)
ECFP (Em)
ECFP (Ex)
Spectral o)erlap
decreasein #onor>mission
increase in Acce/tor>mission
rster #istan 8 0 &or >C and >=: ca. 5 nm 50P8> >&&iien3: no 8>7Signal "eyond 10 nm.
8> can "e a//lied to ,isualie the interaction
o& signaling molecules in li,ing cells
253
Overview of FRET-Microscopy Techniques
1. Acuisition with 8> &ilter set donor ecitation and acce/toremission3: ro"lem: coecitation o& the acce/tor at the donorwa,elength &alse /ositi,es
2. Acuisition o& a ratio image o& acce/tor &luorescence at donorecitation and donor &luorescence at donor ecitation27ilter 8> Microsco/y: Just wor%s when there is eual e/ression
o& donor and acce/tor e.g. in &usion /rotein* "iosensors3
4. 47ilter 8> Microsco/y
6. #etermine the %inetics o& donor &luorescence "leaching this isslower in the /resence o& a 8> acce/tor* as /art o& the "leachingenergy is trans&ered to the acce/tor3
5. #onor reco,ery a&ter acce/tor /hoto"leaching
. &luorescence li&etime microsco/yD $IMF &luorescence li&etime o& thedonor decreases in the /resence o& a 8> acce/tor
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ECFP and EYFP-Scans
0.0
0.2
0.4
0.6
0.8
1.0
1.2
340 360 380 400 420 440 460 480 500 520 540 560 580 600nm
r e l a t i v e
f l u o r e s c e n c e
S/ectral crosstal% o& donor and acce/tor
ro+lems:
1. Co-excitation of t%e acceptor at t%e "onor-excitation ,a)elengt% H#on-0!ET-0luorescence in t%e ra,-0!ET c%annel
2. Signal-o)erlap of donor into t%e acceptor c%annel H #on-0!ETfluorescence in t%e ra,-0!ET c%annel
>citation window
o& donor
>mission window
o& acce/tor
raw 8>7channel:
#onor >citation G Acce/tor >mission
255
8atio o& donor emission and acce/tor emission at the ecitationwa,elength o& the donor
ECFP and EYFP-Scans
0.0
0.2
0.4
0.6
0.8
1.0
1.2
350 400 450 500 550 600nm
r e l a t i v e
f l u o r e s c e n c
e
EYFP (Em)
EYFP (Ex)
ECFP (Em)
ECFP (Ex)
excitation
emission-1 emission-2
$imitations:concentration de/endentdonor and acce/tor ha,e to
colocalie com/letely
!ust use&ul &or 8>7"iosensors with co,alentlin%age "etween donor andacce/toreual7molar e/ression and100P colocaliation3
image \ >mission2 : >mission1
2 ilter78> Microsco/y 8atio Imaging3
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256
4 Images are ta%en under constant camera settings3:1. #onor e.g. C7ecitation and emission3*
2. Acce/tor e.g.=7ecitation and emission D this signal is not a&&ected "y 8>
4. 8>7ilter raw 8>: C7ecitation and =7emission3.
A normalied 8> signal image3 can "e calculated "y using correction &actorso"tained with single stained sam/les:
FRETc = IFRET - corrCFP x ICFP – corrYFP x IYFP
corrC0 : ca. 0.59
corr F0 : ca. 0.18
C = neg. control C7= /os. control
47ilter 8> Microsco/y
8> microsco/y eam/le
257
sample "onorc%annel
Acceptorc%annel
0!ETc%annel
corr.factor
corrected0!ET
C alone 100 0 0 0. 0
= alone 0 100 20 0.2 0
non7"oundC G =
100 100 ?0 0
"ound C7= 100 100 10 ?0
corrected FRET = IFRET - corrCFP x ICFP – corrYFP x IYFP
#onor Acce/tor corrected 8> normalied 8>
9ormalied 8>normalied to di&&.e/ression le,els3:
=
×
neg.control
sam/le
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; this is slowed down in /resence o& a 8> acce/tor
time series of images
EYFPECFP
436 nm
FRETECFP
436 nm
time series of images
476 nm 476 nm
C7rotein alone
C7 and =7rotein
8> Microsco/y "y analying the %inetics o&
donor "leaching
259
offset.A0 += −kt e y
single exponential decay
y... Fluor. Signal
A0... starting signal
k... decay constant
t... time
offset... final value
Fluorescencehalf time Tau:τ
= 0.69/k
FRETeff . E = 1 - (τ without / τ with Akzeptor.)
ro"e mit 8>
ro"e ohne 8>
advantages:
concentration independent
donor and acceptor don‘t have to
colocalize completely
Limitation: requires external control,
difficult to obtain a FRET-image
#onor7"leaching%inetics
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#onor reco,ery a&ter acce/tor "leaching: An image o& the donor in the /resence o&the acce/tor is ta%en* then the acce/toris "leached /artially3* &ollowed "yacuisition o& a second donor image
AcceptorDonor
AcceptorDonorFRET
8> Microsco/y "y acce/tor "leaching and
monitoring donor reco,erydo not use &or C =3
261
isualisation o& "iochemical reactions "y 8>microsco/y e.g. /hos/horylations3
#etection o& the auto7/hos/horylation o& >(7rece/tor on yrosine residues using(7>(8 &usion /rotein and Cy47la"eled anti77yr anti"odies
donor reco,ery a&ter acce/tor /hoto"leaching techniue3
GFP
erb2 -P
Cy3
( 8atio image
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$IM: luorescence $i&etime Imaging Microsco/y
he li&etime o& donor &luorescence usually in the nanosec. range3 isreduced in /resence o& a 8> acce/tor.
his li&etime can "e determined "y a s/ecial ,ariant o& microsco/y. +sually a/ulsed or a modulated laser is used &or ecitation. he &luorescence decayime #omain3 or the /hase shi&t reuency #omain3 o& the emissioncom/ared to the ecitation is a measure o& the &luorescence li&etime.
$IM image&luor. image
263
8>7Biosensors I
Cas/ase 47Biosensor:
A/o/tosis Acti,ation o& cas/ase 43 is detected with a C7= &usion/rotein in which C and = are se/arated "y a cas/ase 4 clea,age site.
Lithout a/o/tosis: 8>* with a/o/tosis: no 8>
7;#>#;7 8>
no 8>
Cas/ase 4C =
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-ther eam/les &or 8>7Biosensors
KA Acti,ity sensor: C and = se/arated "y a KA7su"strate seuenceand a 167474 domain* which "inds /hos/ho7serine o& the KA su"strate
domain.
C =
8>
=CKA
Calcium7Biosensor: Ca2G7sensiti,e Calmodulin and a Ca2G Calmodulin7"indingM14 domain are s/liced "etween C and = additional localiationseuences can "e added D e.g. signal /e/tide and >8 retention seuence3. Achange in the calcium concentration leads to a change in the con&ormation o&the lin%er and thus to an alteration o& the 8> signal.
PKA-substrate 14-3-3
C =Ca2G
Calmodulin M14Ca2G
C
=8>
265
#igital Image Analyses
-&ten used: 'mageI scienti&ic reeware: htt/:rs"we".nih.go,i! 3
#i&&erent o/erations can "e /er&ormed: contrast enhancement*smoothing* "ac%ground su"traction* measurement o& &luorescence
intensities; D and &urther more e,en math with images can "e done e.g.di,iding one image "y another oneF each /iel ,alue is di,ided "ythe corres/onding /iel ,alue o& the second /icture D at the same/iel coordinates3
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266
#i,ision o& imageswith ImageJ
i8> lugin &or ImageJ
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8> analysis with sel&7written ImageJ macro
neg. control
sam/le
0
2
6
?
10
12
16
9egati,eControl
IKK1GMyc IKK2G Mycsam/le 1 sam/le 2
269
In Situ Ey"ridisation ISE3
htt/:www.cytochemistry.netIn]situ.htm
htt/:osiris.rutgers.edusmmin]situ]hy"ridiation.htm
; &or s/eci&ic detection o& #9A or m89A seuences. A la"eled #9A7 or 89A is hy"ridied to the target seuence in situin the cell or the tissue3 and detected
A//lications:
1. #etection or semi73 uanti&ication o& m89A
2. #etection o& #9A7seuences in chromosomes e.g. translocations*mutations* loss o& genes;3
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rinci/le o& ISE
- a labeled“ probe (e.g. DNA, labeled with Biotin-dUTP by Nick-Translation or anoligonucleotide, labeled by terminal deoxynucleotidyl transferase, TdT) diffusesinto the cell and hybridizes with the target sequence. Addition of formamide to thehybridization buffer lowers the specific hybridization temperature, so that at 37°Conly specific target sequences are bound. The probe is then detected viafluorescence (fluorescence in situ hybr., FISH) or by enzyme activity (e.g. HRPand colour reaction).
IC
271
ISE: Ad,antages and #isad,antages o& ,arious /ro"es
Probe type Advantages Disadvantages
DNA (double strand) Easy to use
Subcloning unnecessary
Choice of labeling methods
High specific activity
Possibility of signal amplification
(networking)
Reannealing during hybridization
(decreased probe availability)
Probe denaturation required,
increasing probe length and
decreasing tissue penetration
Hybrids less stable than RNA probes
DNA (single strand) No probe denaturation needed
No reannealing during hybridization
(single strand)
Technically complex
Subcloning required
Hybrids less stable than RNA probes
RNA Stable hybrids (RNA-RNA)
High specific activity
No probe denaturation needed
No reannealing
Unhybridized probe enzymatically
destroyed, sparing hybrid
Subcloning needed
Less tissue penetration
Oligonucleotide No cloning or molecular biology
expertise required
Stable
Good tissue penetration (small size)
Constructed according to recipe
from amino acid data
No self-hybridization
Limited labeling methods
Lower specific activity, so less
sensitive
Dependent on published sequences
Less stable hybrids
Access to DNA synthesizer needed
more recently: BAC /ro"es can "e o"tained &rom collections3: high sensiti,ity &or single co/y genes;
&rom: eldman* 8S* Meyer* JS* and Wuener* $ 1@@<3. rinci/les o& 9euro/sycho/harmacology . Sunderland* MA: Sinauer Associates* Inc. ages 41745.
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>am/les &or ISE
luorescence In Situ7Ey"ridiation onMeta/hase7chromosomes
#etection o& a target7m89A in cryo7or /ara&&in sections
"etection ,it% 55
"etection ,it% alkal. %osp%atase
ISE with inter/hase nuclei
273
m89A7ISE rotocol
Cells are fixed ,it% fres%ly made 6J formalde%yde in GS* p8 .6 for 1 min atroom temperature. All solutions s%ould +e made in 4olecular Giology gradeultrapure ,ater &no !#ase(. Kear glo)es at all times and use sterile disposa+lepipets and tips.
After rinsing in GS &5 L 1/ min. eac%(* cells are permea+ilied ,it% /.J Triton L-1// in 1L GS for 1/ min at 6oC.
Cells are t%en rinsed in GS &5 L 1/ min. eac%( and t%en in 2L SSC &1 L min.(. 1// ng of nick translated pro+e &containing digoxigenin d;T( and 2/ ug of
competitor E. coli t!#A per co)erslip are dried do,n in a Speed 9ac &Sa)ant(.T%is is a good starting place +ut you may %a)e to titrate your specific pro+e.
1/ Ml of deionied formamide is added to t%e dried "#A.
T%e pro+e and t!#A are denatured +y %eating for 1/ min at N/oC. T%e pro+e isc%illed on ice immediately. 1/ Ml of 8y+ridiation +uffer &2/J dextran sulfate 6L SSC( is added to t%e
denatured pro+e so t%at t%e final concentrations in t%e %y+ridiation mixtureare ngml of pro+e* 1 ugml of E. coli t!#A* 2L SSC* and 1/J dextran sulfate.
2/ Ml of %y+ridiation mixturepro+e is placed onto eac% co)erslip. Co)erslips are in)erted onto a slide and sealed ,it% ru++er cement and
incu+ated in a %umid c%am+er for 1 %rs. at 5oC. After rinsing in 2L SSC/J formamide at 5oC* 2L SSC and 1L SSC at room
temperature for 5/ min. eac%* t%e co)erslip containing cells are incu+ated in 6LSSC/.2J GSA2ugml anti-digoxigenin anti+ody for / min. in a %umidc%am+er at room temperature in t%e dark.
Co)erslips are t%en rinsed in 6L SSC &1 L 1 min.( at room temperature* 6LSSC/.1J Triton L-1// &1 L 1 min.(* and 6L SSC &5 L 1/ min. eac%(.
Co)erslips are mounted in fluorescence mounting medium.
4odified from: Iimne-=arcPa* $. and ".$. Spector. 1NN5. Cell 5* 6-N.
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Su/erresolution Microsco/y I
S>#: Stimulated >mission #e/letion
274
A second laser de/letion laser3 Htrims the ecitation s/ot /oint7s/read &unction* S3 to asmaller sie. 8esolution a//r. ?0 nm.
htt/:eiss7cam/us.magnet.&su.eduarticlessu/erresolutionintroduction.html
Su/erresolution Microsco/y IIStructured Illumination Microsco/y SIM3
275
A second laser de/letion laser3 Htrims the ecitation s/ot /oint7s/read &unction* S3 to asmaller sie. 8esolution a//r. ?0 7 100 nm.
A %nown /attern is /ro!ected into theimage /lane at di&&erent angles andinter&eres with sam/le structures*creating Moirj /attern. Su/erresolutionin&ormation can now "e ca/tured "y themicrosco/e &rom these structures "ymathematical algorithms.&rom www.eiss.de3
normal image SIM image
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Su/erresolution Microsco/y
7 "y single molecule detection
276
STO!4: Stochastic O/tical ! econstruction 4icrosco/y using single &luorescent molecules
A$4: hotoacti,ated $ocaliation 4icrosco/y
8esolution: a//r. 40 nm* "ased on statistical calculation o& the center o& a (aussian it o& asingle molecule. 8euires a sensiti,e camera e.g. >MCC#: >lectron7multi/lying charge7cou/led de,ice cameras3 D and some so&tware* "ut no s/eci&ic hardware
htt/:eiss7cam/us.magnet.&su.eduarticlessu/erresolutionintroduction.html
277
8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions &luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
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0lo, CytometrySome contents are ins/ired "y luorescence S/ectrosco/y in
Biological 8esearchX
"y 8o"ert . Mur/hy
0lo, Cytometry D Measuring /ro/erties o& cells in &low
0lo, Sorting D Sorting se/arating3 cells "ased on /ro/erties measured in
&low
D Also called 0luorescence-Acti)ated Cell Sorting &0ACS(
#e&initions:
279
Basics o& low CytometryBasics o& low Cytometry
•Cells in suspension
•flow in single-file
through
•an illuminated volume
where they
•scatter light and emit
fluorescence
•that is collected,
filtered and
•converted to digital
values
•that are stored on a
computer
Fluidics
Optics
Electronics7 low Cytometry* low Analysis3.
7 low Sorting* luorescence Acti,ated Cell Sorting* ACS
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280
htt/:/ro"es.in,itrogen.comresourceseducation
281
9eed to ha,e cells in sus/ension &low in single&ile through an illuminated ,olume
In most instruments* accom/lished "y
in!ecting sam/le into a s%eat% fluid as it/asses through a small 507400 Qm3 orifice
luidics
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Lhether &low will "e laminar can "e determined &romthe !eynolds num+er
282
luidics II
Lhen conditions are right* sam/le &luid &lows in a central corethat does not mi with the sheath &luid
his is termed $aminar flo,
is the mean &luid ,elocity in SI units: ms3
# is the diameter m3
_ is the dynamic ,iscosity o& the &luid aks or9ksm3
is the %inematic ,iscosity \ _ 3 ms3
is the density o& the &luid %gm3
W is the ,olumetric &low rate ms3
A is the /i/e cross7sectional area m3
Lhen 8e 2400* &low is alwayslaminar
Lhen 8e 2400* &low can "etur"ulent
283
Flow Cell
InjectorTip
Fluorescencesignals
Focused laserbeam
Sheathfluid
Purdue University Cytometry Laboratories
8ydrodynamic0ocusing
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The figure shows the
mapping between the
flow lines outside and
inside of a narrow
tube as fluid
undergoes laminar
flow (from left to
right). The fluid
passing through crosssection A outside the
tube is focused to
cross section a inside.
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Eydrodynamic ocusing
285
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Notice how the ink is focused
into a tight stream as it is drawn
into the tube under laminar
flow conditions.
Eydrodynamic ocusing II
Sam/le container and the sheath &luid container are /ut under de&ined air/ressure. $aminar &low is maintained and hydrodynamic &ocussing isachie,ed3 "y a /recise control o& the /ressure di&&erence "etween the sam/lecontainer* the sheath &luid container and the atmos/here
>am/le: ocusing o& in% "ylaminar &low into a ca/illary
In &low cytometry:
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H.B. Steen - MLM Chapt. 2
Flow
through
cuvette
(sense inquartz)
low Cham"er
#i&&erent ty/es are /ossi"le*
a commonly used ty/e:
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PMT
PMT
PMT
PMT
DichroicFilters
Bandpass Filters
Laser
1
2
3
4
Flow cell
original from Purdue University Cytometry Laboratories;
modified by R.F. Murphy and J. Schmid
SSC
FSC
-/tics: (eneral scheme
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orward Scatter SC3Lhen a laser light source isused* the amount o& lightscattered in the &orward
direction along the same aisthat the laser light is tra,eling3
is detected in the for,ardscatter c%annel
he intensity o& &orwardscatter is mainly /ro/ortional
to the sie and sur&ace
/ro/erties o& cells or other/articles3
289
;sually not a sensiti)e 4T detector&+ecause t%e lig%t intensity is %ig%(
Q +ut rat%er a p%otodiode detector*,%ic% can +e set in log1/-increments &E-1* E/* E1...(* and alinearity gain factor &1./ Q N.NN(.
A +locking +ar pre)ents t%e directlaser lig%t from %itting t%e detector.
A predefined 0SC-Kert is often usedas t%res%old to discriminate+et,een cells and dust particles
orward Scatter#etector
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orward scatter threshold to ignore de"ris andsmall /articles or cells
290
291
Lhen a laser light source isused* the amount o& lightscattered to the side/er/endicular to the ais thatthe laser light is tra,eling3 isdetected in the side or N/o
scatter c%annel
he intensity o& side scatter ismainly de/endent onsu"cellular structures e.g.granules* ,esicles;3
Side ScatterSSC3
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90 Degree Light Scatter (SSC)
FSC Detector
SSC detector
Laser
Purdue University Cytometry Laboratories
N/° scattered lig%t&side scatter( isdetected ,it% ap%otomultiplierdetector* ,%ere t%esensiti)ity can +e setin 9olt. Amplificationscale can +e linear orlogarit%mic.
-/tics: luorescence Channels
dichroic mirrors
"and/ass &ilters
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Ethidium
PE
cis-Parinaric acid
Texas Red
PE-TR Conj.
PI
FITC
600 nm300 nm 500 nm 700 nm400 nm
457350 514 610 632 488 CommonLaserLines
Purdue University Cytometry Laboratories
luoro/hores
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Sorting o& cells
488 nm Laser
+-chargedelectrodes
Sortedcells
Fluorescence
FSCSensor
+ - control unit
-
Purdue University Cytometry Laboratories; modified
GatingFSC
FL1
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296
297
Sorting Mode
>clusion7Mode: dro/lets containing non target cells are dismissede,en i& they contain target cells high /urity* may"e lower yield
8eco,ery7Mode: dro/lets containing target cells are collected* e,en i&they also contain non7target cells high yield* may"e low /urity.
Single Cell Mode: only dro/lets with single target cells are collected
high /urity* may"e low yield* high counting accuracy.
I& there are 2 or more cells in one dro/let*and the dro/let contains target and nontarget cells* then there is a Hsorting con&lict*which has to "e sol,ed "y de&ining ana//ro/riate sorting mode:
arget7Cell
9on7target cell
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298
-,erla/ o& &luorescence signals and HCom/ensation
299
HCom/ensationLhen cells are la"eled with 2 &luoro/hores e.g. IC and>3* there might "e a signal crosstal% "etween the detectionchannels de/endent on detector ,oltage settings3. his can"e com/ensated in the &ollwing way: measuring a sam/lecontaining only &luoro/hore 1: i& there is crosstal%* you seean ele,ated intensity in "oth channels D using thecom/ensation control o& the so&tware* you can "ring the&luorescence signal in the wrong channel down to the"ac%ground &luorescence. Lith a sam/le stained only with&luoro/hore 2 you do the same &or the other channel. hiscom/ensation is usually !ust ,alid &or the detector settingsat which it was set.
com/ensation &or &luoro/hore 1com/ensation &or &luoro/hore 2
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300
Com/ensation II
"e&orecom/ensation
a&tercom/ensation
true dou"le /ositi,es can"e determined
301
ACS7(ra/hs
Eistograms: the intensity o& onechannel is di,ided into classeso&ten 1026* 7ais3 and the&reuency o& He,ents cells3scored into these 1026 classes.
#ot lots: 2 di&&erent /arameters
are /lotted on the 7 and y7aisFeach cell is a s/ot on this gra/haccording to its /arameterintensities.
counts
0
$1
$2
Correlation "etweenhistograms anddot/lots
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ACS7(ra/hs II
4#7lots: he &reuency o& e,entsmeasured &or 2 /arameters is illustrated"y a third ais 7ais3 in a 4# manner.
Contour lots: he &reuency o& e,entscells3 in ,arious areas o& a 2#7/lot is,isualied "y lines re/resenting eual/ro"a"ility similar to contour lines o&mountains on a ma/3
$1
$2
$1
$2
#ensity lots: he &reuency o& e,entscells3 in ,arious areas o& a dot /lot iscolour coded to highlight the /ea%s
$2
303
Statistics and H(ating
8egions: can "e de&ined indi&&erent &orms /olygons*elli/ses..3. hese can "e used &orstatistics o& cells &alling into acertain region D "ut also &or Hgating that means re!ectingcertain cells inside or outside3 acertain region &or analysis or
data acuisition3 D or &or instance&or sorting o& cells.
Mar%er: +//er and lower limits inhistograms &or uanti&ication
Wuadrants: s/lit the 2# gra/h in 6regions* "y the coordinates o& the/oint where 2 ais intersect. Allows &ast and sim/le statistics.
8egion 81: @.4P
olygon78egion3
Mar%er M1: <.5P
+$ u//er le&t3: 0.0P+8 u//er right3: 2@. ??P$$ lower le&t3: 0.6P$8 lower right3: @.66P
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8esearch Methods 7 -,er,iew
cell culture systems
la"elling and trans&ection o& cells
analyses o& cellular com/onents
analyses o& molecular interactions
&luorescence measurements
microsco/y
&low analysis ACS3
analyses o& cellular /rocesses /roli&eration* a/o/tosis..3
305
Analytical A//lications o& low Analysis
$eu%ocyte analyses
henoty/ing o& cells C#7Mar%er3
Immuno&luorescence stainings
cell cycle analyses #9A7content3
Chromosome analyses
roli&eration assays Brd7+ incor/oration3
A/o/otosis assays Annein * d* JC13
Calcium &lu7measurements
etc.
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$eu%ocyte analyseshenoty/ing o& cells
C#?7/ositi,e 7cells
C#67/ositi,e 7cells
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cell cycle analyses "y staining o& #9A with I
10000 200 400 600 800
G0G1
s G2M
DNA Gehalt2N 4N
G2M
G1
s
G0
a/o/toticcells
(0 (1
(2 M
S
ropidium-'odide 0luorescence of permea+ilied cells after digestion of !#A:fluor. depends on "#A content
di/loidChromosome content
dou"led di/loid
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rotocol o& a ro/idium7Iodide Staining
Adherent cells:trypsinized, suspended in medium + 10% FCS, centrifuged (1000 rpm, 5 min), Pellet
suspended in PBS (1 ml)
Suspension cells:
Centrifuged (1000 rpm, 5 min), Pellet suspended in PBS (1 ml)
Fixation with EtOH:Pipet cell suspension into 2.5 ml absolute EtOH (final concentration approx. 70%) - or vortex
the suspension at half speed while adding the EtOH) – to prevent clustering of cells during thefixation. Incubate on ice for 15 min (or over night at –20°C).
Alternative fixation with paraformaldehyde:
Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.
Staining:
Pellet the cells at 1500 rpm for 5 min, Suspend the pellet in 500 µl PI-solution in PBS:
50 µg/ml PI from 50x stock solution (2.5 mg/ml), 0.1 mg/ml RNase A, 0.05% Tritin X-100Incubate for 40 min at 37°C
Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant
Suspend the pellet in 500 µl PBS for flow analysis
(you can also leave about 500 µl of the diluted staining solution on the pellet and suspend the
cells in this solution > less loss of cells when you take off the sup.) – the rest of the stainingsolution does not interfere with the flow analysis.
Flow analysis:
Approximate settings (on FACSort):FL1: 570 V log. (e.g. if you want to detect GFP)
FL2: 470 V linear
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Chromosome Analyses
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Analysis o& roli&eration
Brd+7la"eling o& S7hase Cells3
IC7anti7Brd+ luor.
ro/idium!odid luor. I3
Cells are cultured &or a gi,entime in medium containingBromodeoy7+ridin Brd+3. hisnucleotide analogon incororatesinto newly synthesied #9A o&cells in S7/hase3 D and can "edetected with anti7Brd+anti"odies e.g. IC la"eled3.
311
Staining o& a/o/totic cells with JC71
JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide)
is a dye, which incorporates in to mitochondrial membranes, where the fluorescence
depends on the membrane potential !n normal cells (with intact mitochondrial
membrane potential) it builds a""re"ates and emits mainly red fluorescence, in
apoptotic cells (where the membrane potential brea#s down) it occurs in monomers
and emits mainly "reen fluorescence $his can be detected by flow analysis or
microscopy
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rotocol o& a JC71 Staining o& A/o/totic Cells
JC71 is /re/ared as a 1000 stoc% solution in #MS- 5 mgml3.
or the staining o& adherent cells it is diluted in medium to 5 Qgml with,orteing during the dilution to /re,ent the &ormation o& /reci/itates3F the JC71 containing medium is added to the cells* &ollowed "y incu"ation &or 10 minat 4<°C or 8 &or 15 min3.
Su"seuently the cells are washed twice with BS* try/sinied* sus/ended in500 Ql BS and analyed "y &low analysis.
Sus/ension cells lym/hocytes3: sus/end 1:1 with 10 Qgml JC71 in medium&inal conc.: 5 Qgml3
A//roimate detection settings on ACSort:
$1: 40 log3
$2: 410 log3
Com/ensation : $17<P $2 und $27<6P $1
313
#etection o& a/o/totic cells "y I7staining o&/ermea"ilied cells cell cycle analysis3
ragmented #9A emitslower &luorescence thencells with the normaldi/loid #9A content D this
HSu"7(0(1 /o/ulationre&lects a/o/totic cellswith &ragmented #9A 3ata late stage o& a/o/tosis3
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#etection o& a/o/totic cells with Annein
luorescence7la"eled Annein : "inds to/hos/hatidylserine* whichis normally on the innerlea&let o& the mem"rane*"ut which is &li//ed to theoutside during a/o/tosis
#iscrimination "etween necrotic andapoptotic cells
I7/osAnnein7neg.: necroticI7negAnnein7/os.: early a/o/toticI7/osAnnein7/os.: late a/o/totic* ornecrotic with large holes in the mem"ranewhere Annein can get through3
315
Calcium lu #etermination "y low Analysis
Cells are stained with a calcium sensiti,e &luoro/hore I9#-71 or"etter luo63F a&ter a stimulus* the %inetics o& the &luorescence signalis measured monitoring calcium in&lu3.
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Sources
urdue ;ni). Cytometry $a+oratorieshtt/:www.cyto./urdue.edu
8esearch Institute o& Scri//s Clinic: htt/:&acs.scri//s.edu
In,itrogen utorialshtt/:www.in,itrogen.comsiteusenhomesu//ortutorials.html3
B# utorials: htt/:www."d"iosciences.comsu//orttrainingit&]launch.!s/
I. aul !o+inson urdue +ni,.3
!o+ert 0. 4urp%y Carnegie Mellon +ni,.* itts"urgh3
0lo, Cytometry and Sorting* 2nd ed. M.8. Melamed* .$indmo* M.$. Mendelsohn* eds.3* Liley7$iss* 9ew =or%* 1@@0 D
a"ge%'rt: 4$4 0lo, Cytometry: 'nstrumentation and "ata Analysis
M.A. an #illa* .9. #ean* -.#. $aerum* M.8. Melamed* eds.3* Academic ress* $ondon* 1@?5 D a"ge%'rt: 9"$4
317
Com/anies o&&ering &low analysis eui/ment
Becton7#ic%insonhtt/:www."d.com
Bec%man7Coulterhtt/:www."ec%mancoulter.com
Milli/ore:htt/:www.milli/ore.com
Accuri now /art o& B#3htt/:www.accuricytometers.com
artec: htt/:www./artec.com
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318
ree So&tware: lowing So&tware 2htt/:www.&lowingso&tware.com
lowing So&tware 2
319
8egions in histograms
Wuadrants
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320
Cell cycle analysis with lowing So&tware
321
• #e&ine 4 histogram regions E1* E2* E4:(0(1* S and (2M7/hase* res/ecti,ely3
• Acti,ate the region control tool: Cell Cycle
• #e&ine the (2 /ea% multi/lier and /ea% widthright clic% in the cell cycle window3
• Choose acti,e control a&ter right clic%ing3
• Create statistics "y right clic%ing into thehistogram window3
• Create a Stat.$ist "y right7clic%ing into theStatistics window
• Ctrl79 loads the net &ile* ad!usts the E17E4regions automatically and calculates the cellcycle /hases E17E43
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issue Cytometry: Wuantitati,e Image Analysis o&
microsco/y sam/les
htt/:www.tissuegnostics.com
Single7cell recognition e.g. "ased on #AI7nuclear &luorescence3 generation o& cell mas%s &oruanti&ication o& signals e.g. in di&&erent &luorescence channels3 scattergrams can "e deri,ed similar to&low cytometry &or the cells in their original tissue en,ironmentU3 (ating and statistics are /ossi"le
Similar e,aluations can "e done withImageJ using automatic threshold and the HAnalye /articles &eature.
issue Arrays &or uantitati,e com/arison o& sam/les
CA PINNormal
Pat. #1
Pat. #2Pat. #3
Pat. #4Control
Gingiva
Cystectomy
Example of a
prostate tissuearray
> equal staining conditions for all samples
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Multi7/arallel coordinate /lots &or ,isualiation
o& se,eral /arameters in /arallelCan "e done with reeware Mondrian:
htt/:www.theusrus.deMondrianinde.html3
-ne e,ent e.g. one cell3 is re/resented "y a line lin%ingse,eral y7ais &or the di&&erent /arameters e.g.&luorescence signals3F a H/o/ulation can "e selectedand is highlighted also &or the other /arameters. hedata density can "e reduced using a so called al/ha7&actor3 to o"tain "etter ,isi"ility o& numerous data/oints.
he density o& lines e,ents3 can "e ,isualied in colour7coded manner eam/le: 107/arameter /arallelcoordinate /lot generated with Mat$a"3 &ast intuiti,e,isualiation o& multi/arameter data sets
1 2 4 6 5 < ? @ 10