bio 6 – sds-page lab - sds... · a polyacrylamide gel that is subjected to electric current. the...
TRANSCRIPT
Bio6–SDS-PAGELab
ObjectivesUponcompletionofthislaboratoryyouwillunderstandhowtoloadandrunproteinsamplesonanSDS-polyacrylamidegel,stainthegel,andanalyzetheresultingbandsofproteinonthegeltoestimatethemolecularweightofeachprotein.
Introduction
SDS-PAGE is a very common laboratory technique used to analyze proteins. The acronym SDS-PAGEstandsforsodiumdodecylsulfate–polyacrylamidegelelectrophoresis.SodiumdodecylsulfateorSDSisadetergentcommonlyusedinbiologylaboratoriestodenatureproteins,i.e.,disruptthe3-dimensionalstructureofproteinswithoutbreakingthepolypeptidebackbone.Polyacrylamideisapolymerpreparedasagelatinousmediumor“gel”throughwhichproteinscanberesolvedbasedonmolecularweight(MW).Electrophoresisreferstothemovementofchargedsolubleparticlessuchasproteinsthroughamediumduringexposuretoanelectricfield.SDS-PAGEisthereforeatechniquebywhichproteinsmovethroughapolyacrylamidegelthatissubjectedtoelectriccurrent.The rate at which a protein moves through the microscopic pores of a polyacrylamide gel duringelectrophoresisisdependentonthreephysicalproperties–molecularweight,3-dimensionalshape,andnetcharge.SeparationofproteinsbasedsolelyonthepropertyofMWispossibleonlyifthevariablesof3-dimensionalshapeandnetchargeareeliminated.ThisisaccomplishedbythedetergentSDS,whichduetoitsamphiphilicpropertiesofbeinghydrophobicononeendandchargedontheotherisabletodisruptallnon-covalentinteractionsinaprotein–i.e.,denaturetheprotein.Thisresultsinunfoldedorlinearpolypeptidesthathaveanetnegativelychargeasshownbelow:
SDS thuseliminatesanydifferences in shapeandoverall chargeamongproteins, leavingonevariablephysicalproperty–molecularweight–toinfluencetherateatwhichaproteinmovesthroughthegel.
57
Asshownbelow,SDS-PAGEisrunverticallyandthegelconsistsofa“stackinggel”ontopofa“resolvinggel”(aka“runninggel”).Bothendsofthegelareincontactwithanelectrolytebuffersolutioncontainingthepositiveandnegativeelectrodeswhichgenerateelectriccurrentthroughthegel.
Thestackinggelcontainscavitiescalledwells intowhichyouwill loadproteinsamples. Whenelectriccurrentisapplied,negativelychargedsolutessuchasSDS-treatedproteinswillexperienceelectromotiveforcetowardtheoppositelychargedpositiveelectrodeandthusmoveintothestackinggel.ThestackinggelhasanacrylamidepercentageandpHthatensuresallproteinsentertheresolvinggelatthesametime.Onceintheresolvinggel,proteinswillmigrateatarateinverselyproportionaltoMW.Whenthegelisfinishedrunning,itisstainedtorevealthepositionofeachproteinonthegelasshownbelow:
+
–
58
Eachstainedbandonthegelcorrespondstoaoneormoretypesofproteinwith thesamemolecularweight(keepinmindthateachbandcontainsbillionsofproteinsofthatMW).Thelargertheprotein,thesloweritmovesthroughthegelandthusthehigheritsposition.Smallerproteinsmoveatfasterrateandareseenasbandsfurtherdownthegel.AproteinMWstandard(acollectionofproteinsofknownsize)isalwaysrunonthegelandusedtoestimatethesizesofproteinsintheotherlanes.
Part1:RunninganSDS-PAGEgelTobeginthis labyouwilldenatureandloadseveraldifferentproteinsamplesonapolyacrylamidegelafterwhichyouwillrunthegelasdescribed.PreparinganacrylamidegelforSDS-PAGEisabittricky,sothepolyacrylamidegelshavebeenpreparedforyou.Todenatureyourproteinsitisessentialthatyouaddsample loadingbuffer and thenboil the sample. Thesample loadingbuffer contains severalkeycomponentsneededtofullydenaturetheproteinsandhavethemrunthroughthegelproperly:
• SDStodenaturethepolypeptides• Dithiothreitol(DTT)tobreakdisulfidebonds(covalentbondsbetweencysteines)• Glyceroltomakethesampledenseenoughtosinkintothewell• BromophenolBlue,anegativelychargeddyetomonitorgelprogress
Theloadingdyeispreparedata2Xconcentrationsothatitcanbedilutedto1Xwhenmixedwiththeproteinsample.Onceloadingdyehasbeenadded,theheatfromboilingfacilitatesdenaturationoftheproteinsandbreakingofdisulfidebonds.Disulfidebonds(refertochapter5ofyourtextbook)aretheonlycovalentinteractionsformedbetweenaminoacidRgroupsandarenotdisruptedbySDS,thustheneedforDTT.Onlyinthisfullydenaturedstatewilleachproteinsamplemovethroughthegelataratedependentonmolecularweightonly.Loadingsamplesintothewellsofthegelcanbechallengingsincethegelisverythin,toothintoinsert the pipet tip directly into the well. Asshowninthepicture,youwillneedtogentlyrestthepipettipontopoftheinnerplateatananglesothatthepipettipisrestingjustabovethewellyouwanttoload.Itisespeciallyimportantthatyoudonotpressthepipettipdownbetweentheplates,otherwiseyoumayprythemapart.Ifthishappens,evenjustalittlebit,yoursampleswillleakdownbetweenthegelandtheplateandbelost.Beforeyouloadyourproteinsamplesyouwillloadapracticesamplesoyouwillhavesomeexperiencebeforeloadingyourrealsamples.
59
Exercise1–LoadingandrunningyourgelAllofthematerialsyouwillneedcanbefoundoncartsatthesideofthelabwhereyouwillobtainanicebucketandatrayofmaterials.Onceyouhaveallyourmaterials,besuretohalffillthebeakerwithhottapwaterandputitonahotplatesettothemaximumsoboilingwaterwillbereadywhenyouneedit:
1. Each protein sample is 5 µl. Add 2X sample loading buffer to each protein sample so the finalconcentrationis1X(calculatethisasyoudidinthe“MetricSystem”lab).
2. Tapeachtubegentlytomixandboilthesamplesalongwiththemolecularweightmarkerfor5minutes(theproteinmolecularweightmarkeris5µlandalreadycontains1Xsampleloadingbuffer).
3. Identifythewellsyouplantouseforyourproteinsamples(avoidusingdeformedwells),andindicate
inyourlabnotebookhowyouwillloadyourgel.Eachpersonshouldpracticeloading10µlof1Xsampleloadingbufferintheunusedlanes.Yourinstructorwilldemonstratehowtodothis.
4. Loadalloftheproteinmolecularweightmarkerandeachproteinintotheirdesignatedlanes.
5. Placethecoveronthegelapparatusandconnecttheredandblackplugstothecorrespondingcolors
inthepowersupply.NOTE:Ifthebufferlevelintheupperchamberisnotabovetheinnerplateofthegel,therewillbenoflowofelectriccurrent.
6. Besurethepowersupplyispluggedinandsetthetoggleswitchbelowthedigitaldisplayto“Volts”,
theRangeSelectswitchto“Low”andtheVoltageSelectknobto“Min.”
7. Turnonthepower(toggleswitchonupperleft)andadjusttheVoltageSelectknobuntilthedigitaldisplayreadsbetween110and120volts.NOTE:Ifthegelisrunningproperlyyouwillseeacurtainoftinybubblesrisingfromthewireinthe
upperbufferchamber.Youshouldalsoseeamuchloweramountof“bubbling”fromthewireinthelowerbufferchamber.Ifyoudon’tseethis,besuretoconsultyourinstructor.
8. Letthegelrununtilthebromophenolbluedyeis~1cmfromthebottomofthegel(~90minutes).9. Whentheruniscomplete,turntheVoltageSelectknowto“Min.”andturnoffthepower.
60
Part2:STAININGPROTEINSINAPOLYACRYLAMIDEGEL
TovisualizetheproteinsinyourpolyacrylamidegelyouwilluseastaincalledCoomassieBrilliantBlue.Thisisnottheonlyproteinstainonecoulduse,howeveritisaverycommonlyusedstaintoviewproteinsonpolyacrylamidegels.Thestainingprocessmayrequireanovernightincubationsoyoumaynotseetheproteinbandsonyourgeluntilthenextlaboratorysession.Exercise2–Stainingapolyacrylamidegel
Besuretoweargloveswhenyouhandlethegelandstainingsolutions,andtocleanupasindicatedbyyourinstructor:
1. WhenthegelruniscompletebesuretheVoltageSelectknobissetto“Min.”andthepowerisoff,thenremovethecoverofthegelapparatus.
2. Carrythegelapparatustothesinkanddiscardtherunningbufferfrombothchambersintothesink.
3. Loosentheclampsholdingthegelinplace,carefullyremovetheplatescontainingyourgelandlayitona
flatsurfacewiththetallerouterglassplatefacingup.
4. Usetheplasticspatulasuppliedasawedgetocarefullypryapartthe2plates.Thegelshouldsticktooneplateortheotherasyoudoso.Ifitseemstosticktoboth,askforassistancefromyourinstructor.
5. Place~100mloffixativesolutioninthestainingtrayprovided.
6. Gentlyseparateabottomcornerofthegelfromtheglassplateusingthespatula,carefullypeelthegelofftheplate(it’sOKifthestackinggeltearsoff,youwon’tneedit)andplaceitinthefixativesolution.Ifthegelcurlsup,carefullyunravelitwithyourglovedhanduntilitflattensout.
7. PlaceaProteinInstaStaincard(whichcontainsCoomassieBrilliantBluestain)facedowninthefixativesolutionoverthegel,placethelidonthecontainer(besuretolabelthelidwithyourgroupnumber)andplacethecontainerontheslowrockeratthesideofthelab.Leavefor1to3hoursorovernight.
8. Whentheproteinbandsareclearlyvisible,photographyourgelasindicatedbyyourinstructorbeingsuretoincludearuleralignedwiththetopoftheresolvinggel.Storeanimageofthegelinyourlabnotebook.
Part3:ESTIMATINGTHEMOLECULARWEIGHTOFPROTEINSONAPOLYACRYLAMIDEGEL
Youwillusetheproteinmolecularweightstandardyouranonyourgeltoestimatethemolecularweightsoftheotherproteinsonyourgel.Asmentionedearlier,theproteinmolecularweightstandardisasetofproteins of knownmolecular weight. Since the rate at which a denatured proteinmoves through apolyacrylamidegelduringelectrophoresisdependsonly in itsmolecularweight, youcanestimate themolecularweightofaproteinbasedonitspositiononthegelinrelationtothemolecularweightstandardproteins. Intheory, themostaccuratewaytodothis is tocreateastandardcurveonsemi-loggraphpaperbyplottingmolecularweightindaltons(Da)orkilodaltons(kDa)vsdistancetraveledonthegelincm.Thedistancetraveledreferstothedistancefromthemiddleoftheproteinbandtothetopoftheresolvinggel(notthebottomofthewellinthestackinggel).
61
Thegraphaboveshowsasamplestandardcurveconsistingof4knownproteins(NOTE–thestandardproteinsonyourgelaredifferent,thisisanexample).Oncethestandardcurveisdrawn,themolecularweightofanyproteinonthegelcanbeestimatedusingthedistanceittraveledongel.Forexample,ifaproteinonthegelpertainingtothisstandardcurvetraveled7.4cm,identify7.4cmontheX-axisandmoveverticallytofindthecorrespondingpositiononthestandardcurve.ThemolecularweightontheY-axis that corresponds to this point on the standard curvewill be your estimate. In this example themolecularweightestimatewouldbe~45,000daltonsor45kDa.Whilethismethodcanbeveryprecise,itsaccuracydependsonthequalityofthedistancemeasurementsandtheprecisionofthebestfitcurve.Asimplermethodtoestimatemolecularweightistocomparetheproteinbandofinteresttotheproteinmolecularweightstandardandvisuallyestimate itsmolecularweight. Forexample, ifyourproteinofinterestispositionedapproximatelyhalfwaybetweenstandardproteinsthatare40kDaand50kDa,thenyouwouldestimate45kDa.Ifthebandisjustaboveandveryclosetothe40kDastandardproteinthenyoumightestimate41or42kDa.Whilethismaynotseemveryprecise,itcangiveyouavalueverycloseto that obtainedusing a standard curve and formost purposes this shouldbe sufficient. In thenextexerciseyouwillestimatetheMWofproteinsusingbothmethodsdescribedabove.
(Dal
tons
or D
a)
62
Exercise3–EstimatingproteinmolecularweightNOTE:A12%polyacrylamidegelcaneffectivelyresolveorseparateproteinsbetween20kDaand100kDa,sobesureNOTtoincludemolecularweightstandardproteinsoutsidethisrangeinyourstandardcurve.
1. Placeyourstainedgelonawhitetransilluminatoranduseasmallrulertomeasureincmthedistancefromthemiddleofthebandtothetopoftheresolvinggelforeachmolecularweightstandardprotein*between20kDaand100kDa.Recordthesevaluesinyournotebook.
2. Plotthesevaluesonthesemi-loggraphpaperfoundattheendofthislabanddrawthebestfitlinethroughthepoints.Acopyofthisstandardcurveshouldbeplacedinyourlabnotebook.
3. Foreachproteinsampleloadedonyourgel,identifythemostprominentband(thiswillbetheproteinofinterest,theotherbandsarecontaminants),measurethedistancefromthemiddleofthebandtothetopoftheresolvinggel.Recordthesevaluesinyournotebook.
4. Estimatethemolecularweightofeachproteinusingthestandardcurveasshownonthepreviouspageandrecordinyournotebook.
5. Estimatethemolecularweightofeachproteinbyvisualcomparisonwiththeproteinmolecularweightstandardsandrecordinyournotebook.
6. Answerthestudyquestionsassociatedwiththislab.
NOTE:Forthislaboratoryyouwillturninaformallabreport.*Theproteinmolecularweightstandardmostlikelyusedonyourgelisshownbelow.Ifadifferentproteinmolecularweightstandardwasusedyourinstructorwillprovidethedetails.
63
64
SDS-PAGELabStudyQuestions NAME:1.SDS-PAGEisanacronymreferringto3specificcomponents(SDS,PAandGE).Indicatewhat
SDS-PAGEstandsforandbrieflydescribeeachofthese3components.2.Whatdoesitmeanto“denature”aproteinandwhyisthisimportantforSDS-PAGE?3.DescribehowyoudenaturedtheproteinsyouusedforSDS-PAGE.4.Whatsubstanceinsampleloadingbufferisresponsibleforbreakingdisulfidebonds?5.Diagramadisulfidebondunder“BEFORE”andalsodiagramwhatitshouldlooklikeafter
boilinginthepresenceofthesubstancefromthepreviousquestion(“AFTER”).
BEFORE AFTER6.Whatisthepurposeofloading“proteinmolecularweightstandards”onthegel?
65
7.Listthemolecularweightsofthestandardproteinsrunonyourgelandchecktheonesthatyouusedtomakeyourstandardcurve.
8.Describeeachofthetwomethodsyouusedtoestimateproteinmolecularweight.9.Whydidyoumeasurethedistanceeachproteintraveledintothegelfromthetopofthe
resolvinggelandnotthebottomofthewell?
66