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Section 5.2Purification of Cells and Their Parts

Most animal and plant tissues contain a mixture of cell types. However, an investigator often

wishes to study a pure population of one type of cell. In some cases, cells differ in some physical

property that allows different cell types to be separated. White blood cells (leukocytes and redblood cells (erythrocytes, for instance, have very different densities because erythrocytes have

no nucleus! thus these cells can be separated on the basis of density. "ince most cell types cannot

be differentiated so easily, other cell#separation techni$ues have had to be developed. "imilarly,

it is essential to isolate $uantities of each of the ma%or subcellular organelles to study their

structures and metabolic functions in detail.

Go to:

Flow Cytometry Separates Different Cell Types

&flow cytometercan identify different cells by measuring the light they scatter, or the

fluorescence they emit, as they flow through a laser beam! thus it can sort out cells of a particular

type from a mixture. Indeed, afluorescence-activated cell sorter (FACS),an instrument based on

flow cytometry, can select one cell from thousands of other cells ('igure #)*. 'or example, if

anantibodyspecific to a certain cell#surface molecule is linked to a fluorescent dye, any cell

bearing this molecule will bind the antibody and will then be separated from other cells when it

fluoresces in the '&+". nce sorted from the other cells, the selected cell can be grown in

culture.

Figure 5-21

'luorescence#activated cell sorter ('&+". & concentrated suspension of cells is allowed to react

with a fluorescent antibody or a dye that binds to a particle or molecule such as -&.

/he (more...

/his procedure is commonly used to purify the different types of white blood cells, each

of which bears on its surface one or more distinctive proteins and thus will bind monoclonal

antibodies specific for thatprotein. "uch '&+" separations are more difficult to conduct on

cultured cells or cells from animal tissues, which interact with ad%acent cells and are surrounded

by anextracellular matrix. "amples must be treated with proteases to degrade the extracellular#

matrix proteins and cell#surface proteins that attach cells in tissues to one another! these

proteases usually also degrade the distinctive cell#surface 0marker1 proteins that distinguish one

cell type from another.
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ther uses of flow cytometry include the measurement of a cell2s -&and3&content and

the determinationof its general shape and si4e. /he '&+" can make simultaneous measurements

of the si4e of a cell (from the amount of scattered light and the amount of -& it contains (from

the amount of fluorescence from a -binding dye.

Go to:

Disruption of Cells eleases Their !rganelles and !ther Contents

/he initial step in purifying subcellular structures is to rupture theplasma membraneand thecell

wall, if present. 'irst, the cells are suspended in a solution of appropriatepHand salt content,

usually isotonicsucrose (5.) M or a combination of salts similar in composition to those in the

cell2s interior. Many cells can then be broken by stirring the cell suspension in a high#speed

blender or by exposing it to highfre$uency sound (sonication).6lasma membranes can also be

sheared by special pressuri4ed tissue homogeni4ers in which the cells are forced through a very

narrow space between the plunger and the vessel wall. 7enerally, the cell solution is kept at 5 8+

to best preserve en4ymes and other constituents after their release from the stabili4ing forces of

the cell.

9ecause theplasma membraneis highly permeable to water but poorly permeable to the salts

and other small molecules (solutes within cells, osmotic flowcan be enlisted to help rupture

cells. 3ecall that water flows across a semipermeable membrane, such as the plasma membrane,

from a solution of high water (low solute concentration to one of low water (high solute

concentration until the water concentration on both sides is e$ual. +onse$uently, when cells are

placed in ahypotonicsolution (i.e., one with a lower salt concentration than that of the cell

interior, water flows into the cells ('igure #)). /his osmotic flow causes the cells to swell and

then more easily rupture. +onversely, in ahypertonicsolution (i.e., one with a higher salt

concentration than that of the cell interior, water flows out of cells, causing them to shrink.

When cells are placed in an isotonicsolution (i.e., one with a salt concentration e$ual to that of

the cell interior, there is no net movement of water in or out of cells. 'or this reason,

anisotonicsolution is best for preserving normal cell structure.

Figure 5-22
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3esponse of animal cells to the osmotic strength of the surrounding medium. "odium, potassium,

and chloride ions do not move freely across the cell membrane, but water does. (a When

the (more...

-isrupting the cell produces a mix of suspended cellular components, the homogenate,from

which the desired organelles can be retrieved. 9ecause rat liver contains an abundance of a single

cell type, this tissue has been used in many classic studies of cell organelles. However, the same

isolation principles apply to virtually all cells and tissues, and modifications of these cell#

fractionation techni$ues can be used to separate and purify any desired components.

Go to:

Different !rganelles Can "e Separated #y Centrifugation

In +hapter : we discussed the principles of centrifugation and the uses of centrifugation

techni$ues for separating proteins and nucleic acids. "imilar approaches are used for separatingand purifying the various organelles, which differ in both si4e and density.

Most fractionation procedures begin with differential centrifugationat increasingly higher speeds

('igure #):, also called differential-velocity centrifugation./he different sedimentation rates of

various cellular components make it possible to separate them partially by centrifugation. uclei

and viral particles can sometimes be purified completely by such a procedure. &fter

centrifugation at each speed for an appropriate time, the supernatant is poured off and

centrifuged at higher speed. ;ach pelleted fraction can be resuspended and further separated by

e$uilibrium densitygradient centrifugation (discussed next.

Figure 5-2$

+ell fractionation by differential centrifugation. 7enerally, the cellular homogenate is first

filtered or centrifuged at relatively low speeds to remove unbroken cells. /hen

centrifugation (more...

-ifferential centrifugation does not yield totally pureorganellefractions. ne method for further

purifying fractions isequilibrium density-gradient centrifugation,which separates cellular

components according to their density. /he impure organelle fraction is layered on top of a

solution that contains a gradient of a dense nonionic substance, such as sucrose or glycerol. /he

tube is centrifuged at a high speed (about


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reticulum(density = *.)5 g>cm : separates well from the 7olgi vesicles (density = *.*< g>cm :

and from theplasma membrane(density = *.*) g>cm :. (/he higher density of the rough

endoplasmic reticulum is due largely to the ribosomes bound to it. /his method also works well

for resolving lysosomes, mitochondria, and peroxisomes in the initial mixed fraction obtained by

differential centrifugation ('igure #)


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recently developed techni$ue uses tiny metallic beads coated with specific antibodies. rganelles

that bind to the antibodies, and thus are linked to the metallic beads, are recovered from the

preparation by adhesion to a small magnet on the side of the test tube.

Figure 5-2)

Immunological purification of clathrin#coated vesicles. (a & suspension of membranes from rat

liver is incubated with an antibody specific for clathrin, a protein that coats the outer

surface (more...

&ll cells contain a do4en or more different types of small membrane#limited vesicles of about thesame si4e (5@*55 nm in diameter and density. 9ecause of their similar si4e and density, these

vesicles are difficult to separate from one another by centrifugation techni$ues. Immunological

techni$ues are particularly useful for purifying specific classes of such vesicles. 'at and muscle

cells, for instance, contain a particular glucosetransporter (7AB/


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;$uilibrium density#gradient centrifugation, which separates cellular components

according to their density, can further purify cell fractions obtained by differential

centrifugation.

9ecause the membranesurrounding each type of organellecontains organelle#specific

proteins, immunological techni$ues are very useful in purifying organelles and vesicles,

particularly those that have a similar si4e and density.

Figure 5-22esponse of animal cells to the osmotic strength of the surrounding

medium

"odium, potassium, and chloride ions do not move freely across the cellmembrane,but

water does. (a When the medium isisotonic, there is no net flux of water into or out ofthe cell. (b When the medium ishypotonic, water flows into the cell (red arrow until the

ion concentration inside and outside the cell is the same. Here, the initial cytosolic ion

concentration is twice the extracellular ion concentration, so the cell tends to swell to

twice its original volume, at which point the internal and external ion concentrations are

the same. (c When the medium is hypertonic, water flows out of the cell until the ion

concentration inside and outside the cell is the same. Here, the initial cytosolic ion
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concentration is half the extracellular ion concentration, so the cell is reduced to about

half its original volume.

Figure 5-2$Cell fractionation #y differential centrifugation

7enerally, the cellular homogenate is first filtered or centrifuged at relatively low speeds

to remove unbroken cells. /hen centrifugation of the homogenate at a slightly faster

speed or for a longer duration will selectively pellet thenucleusD the

largest organelle(usually @ *5 Em in diameter. & centrifugal force of ?55 g(?55 times

the force of gravity is necessary to sediment nuclei! this is generated by a typical

centrifuge rotor operating at 55 revolutions per minute (rpm. /he undeposited material

(the supernatant is next centrifuged at a higher speed (*,555gF min, which deposits

themitochondria, chloroplasts, lysosomes,and peroxisomes. & subse$uent centrifugation

in the ultracentrifuge (*55,555gF ?5 min results in deposition of the plasma membrane,

fragments of the endoplasmic reticulum,and large polyribosomes. & force of

*55,555gre$uires about 5,555 rpm in an ultracentrifuge! at this speed, the rotor

chamber is kept in a high vacuum to reduce heating due to friction between air and the

spinning rotor. /he recovery of ribosomal subunits, small polyribosomes, and particles

such as complexes of en4ymes re$uires additional centrifugation at still higher speeds.
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nly the cytosolD the soluble a$ueous portion of the cytoplasmD remains undeposited

after centrifugation at :55,555gfor ) hours.

Figure 5-2%Separation of organelles from rat li,er #y euili#rium density-gradient

centrifugation

'or example, the material deposited as a pellet by centrifugation at *,555g(see'igure

#): can be resuspended and layered on a density gradient composed of layers of

increasingly more dense sucrose solutions in a centrifuge tube. Bnder centrifugation,

each organellemigrates to its appropriate e$uilibrium density and remains there. /o

obtain a good separation of lysosomes from mitochondria, the liver is perfused with a

solution containing a small amount of detergent before the tissue is disrupted. /he

detergent is taken into the cells by endocytosisand transferred to the lysosomes, making

them less dense than they would normally be, thereby affording a 0clean1 separation from

the mitochondria.

http://www.ncbi.nlm.nih.gov/books/NBK21492/

Molisch's Test - Qualitative Test inCarbohydrates

Molisch's test is a test for carbohydrates or compounds which canbe dehydrated to furfural or furfural derivatives in the presence ofthe concentrated sulphuric acid (H!"#$% &urfural is derived from
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the dehydration and rearranement of pentoses and pentosanswhile hydroymethyl is produced from heoses and heosans%The )-naphthol reacts with the cyclic aldehydes to form purplecoloured condensation products%

*lthouh this test will detect compounds other than carbohydrates(i%e%$ lycoproteins+ a neative result indicates the absence ofcarbohydrates%

http://web.knust.edu.gh/oer/pages/index.php

siteid!knustoer"page!#nd$materials"cou!41

,iuret's Test - Qualitative Test in roteins

,iuret's test for peptide bonds is a reaction characterised by aviolet colour upon the addition of copper sulphate to allcompounds with two amide or peptide bonds lin.ed directly orthrouh an intermediate carbon atom% /t is used in the detectionand estimation of proteins and peptides havin more than twoamino-acids%

* pin. to purple0violet colour indicates the presence of proteins% *more intense coloration indicates the presence of more compleproteins%

http://web.knust.edu.gh/oer/pages/index.php

siteid!knustoer"page!#nd$materials"cou!42

%he 'olisch %est

Shows positive test for:
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(ll carboh)drates. 'onosaccharides give a rapid positive test.

*isaccharides and pol)saccharides react slower.

Reactions:

%he test reagent deh)drates pentoses to +orm +ur+ural ,top

reaction- and deh)drates hexoses to +orm h)drox)meth)l

+ur+ural ,bottom reaction-. %he +ur+urals +urther react with

naphthol present in the test reagent to produce a purple

product ,reaction not shown-.

How to perform the test:

%wo ml o+ a sample solution is placed in a test tube. %wo drops

o+ the 'olisch reagent ,a solution o+ napthol in 90 ethanol-is added. %he solution is then poured slowl) into a tube

containing two ml o+ concentrated sul+uric acid so that two

la)ers +orm.

A positive test is indicated by:


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%he +ormation o+ a purple product at the inter+ace o+ the two

la)ers.

a negative test ,le+t- and a positive test ,right-http://www.harpercollege.edu/tm

ps/chm/1/dgodambe/thedisk/carbo/molisch/molisch.htm

%he Biuret %est

Shows positive test for:

Biuret and proteins.

How to perform the test:

repare a solution or suspension o+ the sample b) placing 3.2

g in 1 ml o+ water. %en drops o+ 1. ' Na5 ,a colorless

solution- and 2 drops o+ .1 ' 6u74,a light blue solution- are

added.

A positive test is indicated by:

a deep blue/purple color due to the copper ion complex with

the amide group o+ the protein.
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a negative test ,le+t- and a positive test ,right-http://www.harpercollege.edu/tm

ps/chm/1/dgodambe/thedisk/+ood/biuret/biuret.htm

1.What is diphenylamine ?

Diphenylamine is also known as *iphen)lamine redox8 with its CAS o. 1!!"#$"%8 and it has the

molecular +ormula o+ 612511N and a molecular weight o+ 19.22. %he other properties o+ diphen)lamine

include: *ensit): 1.1 'elting point: 24 ;6 Boiling point: ater

solubilit): 7lightl) soluble. .< g/1 m?. *iphen)lamine is o+ten used +or producing stabili@er o+

explosive and +uel.

!. Diphenylamine test

ne o+ the suppliers o+ diphen)lamine8 5ang@hou 6hem+ar ?td.8 which has more than 2 )earsA

experience +ocused on providing integrated development and manu+acturing service to the

pharmaceutical and #ne chemical industr)8 showed that diphen)lamine was o+ great importance inman) experiments.

=or example8 in the identi#cation o+ *N(8 *N( can be identi#ed chemicall) with the dische

diphenylamine test. ne maor diCerence between *N( and DN( is their sugar: *N( contains

deox)ribose8 whereas DN( contains ribose. %his is what is diphenylamine test +or deox)ribose +or. En

this process8 the reaction between the *ische reagent and 2deox)pentose results in the development

o+ a blue color. %he reaction depends on the conversion o+ the pentose to wh)drox)laevulinic aldeh)de

which then reacts with diphen)lamine to give a a blue colored complex. %he intensit) o+ the blue color

is proportional to the concentration o+ *N(. *ische reagent does not react with the ribose sugar in DN(

and does not +orm a bluecolored complex. Dische diphenylamine test ( chemical test utili@ed to

detect the presence o+ *N( in a substance.

http://product.lookchem.com/item/21/diphen)laminetest.html

&ive 1uestions about 2iphenylamine

osted on !eptember 3+ 456by bb7456
http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/biuret/biuret.htmhttp://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/biuret/biuret.htmhttp://product.lookchem.com/item/251/diphenylamine-test.htmlhttps://bbzblog.wordpress.com/2013/09/27/five-questions-about-diphenylamine/https://bbzblog.wordpress.com/2013/09/27/five-questions-about-diphenylamine/https://bbzblog.wordpress.com/2013/09/27/five-questions-about-diphenylamine/https://bbzblog.wordpress.com/author/bbz2013/http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/biuret/biuret.htmhttp://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/food/biuret/biuret.htmhttp://product.lookchem.com/item/251/diphenylamine-test.htmlhttps://bbzblog.wordpress.com/2013/09/27/five-questions-about-diphenylamine/https://bbzblog.wordpress.com/2013/09/27/five-questions-about-diphenylamine/https://bbzblog.wordpress.com/author/bbz2013/


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1.Diphenylamine test for deoxyribose?

Answer1:

When adding diphenylamine to deoxyribose you will get a dark deep purple color. If you

react diphenylamine with crude DNA you will get a pink!iolet color. DIphenylamine

test is "uantitati!e and the darker the color the greater the concentration of DNA in thesolution

2.What is diphenylamine test for deoxyribose for?

Answer#:

It can be used to test for the presence of DNA in a substance. $he test consists of

diphenylamine%&A' N(.:122-39-4) in acid* which reacts with the deoxyribose in the

DNA to produce a blue color. $he intensity of the blue color is in direct proportion to the

concentration of DNA.

(ne ma+or difference between DNA and ,NA is their sugar: DNA contains deoxyribose*

whereas ,NA contains ribose. DNA can be identified chemically with the Dische

diphenylamine test. $he reaction between the Dische reagent and #deoxypentose

results in the de!elopment of a blue color. $he reaction depends on the con!ersion of

the pentose to whydroxylae!ulinic aldehyde which then reacts with diphenylamine to

gi!e a a blue colored complex. $he intensity of the blue color is proportional to the

concentration of DNA. Dische reagent does not react with the ribose sugar in ,NA and

does not form a bluecolored complex

3.What is responsible for the formation of the absorbed color of DNA with

diphenylamine?

Answer -:

When you heat DNA in acid the #deoxyribose is con!erted to whydroxyle!ulinyl
http://www.guidechem.com/cas-122/122-39-4.htmlhttp://www.guidechem.com/cas-122/122-39-4.htmlhttp://www.guidechem.com/cas-122/122-39-4.html


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aldehyde* which reacts with the compound* diphenylamine* to produce a bluecolored

compound.

4.Why does blue colour appears in diphenylamine test?

Answer :/lue color appears in diphenylamine test because of the reaction of nitrates.

.Detection of DNA with Diphenylamine?

Answer 0:

A diphenylamineindicator is often used for the detection of DNA. Diphenylamine is

an organic compound and classified as an aromatic amine due to the presence of a

benene ring in its chemical makeup. &hemical hydrolysis of DNA must take place when

looking for it using diphenylamine. $he DNA concentration in an organism is measured

by looking for the absorbency rate of the diphenylamine. DNA molecules are the

building blocks of life and contain specific genetic coding for the organism it is

contained in.

I sorted out these "uestions and answers about Diphenylamine* I hope for your help.

Want to learn more information about Diphenylamine* you can access

the guidechem.com.2uidechem.com is +ust a place for you to look for some chemicals.

https://[email protected]/21


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,lac. and white microphotoraph ofFeulgen stained+ intact tic. salivary landsinfected by deer tic.

virus%Hypotrophiedsalivary acinusfilled with amorphous masses of pin.stainin (A&eulen positive$ material

(arrows$% !cale bar A 54 Bm%

Feulgen stainis a stainintechni1ue discovered by


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Classi(cation 6olorimetric method

Analytes entoses

"ials testis a chemical test for the presence of pentoses% /t is named after Manfred ,ial+ German

hysician% The components include orcinol+ hydrochloric acid+ and ferric chloride% * pentose+ if

present+ will be dehydrated to form furfuralwhich then reacts with the orcinol to enerate a colored

substance% The solution will turn bluish and a precipitate may form% The solution shows

twoabsorption bands+ one in the red between &raunhofer lines , and C and the other near the 2

line%5*n estimate of the relevantwavelenthscan be made by referrin to the &raunhofer

linesarticle%

&ompositionedit

,ial's reaent consists of 4%# orcinol+ 44 ml of concentrated hydrochloric acid and 4%? ml of a 54Isolution of ferric chloride% ,ial's test is used to distinuish pentoses from heosesJ this distinction is

based on the color that develops in the presence of orcinol and iron (///$ chloride% &urfural from

pentoses ives a blue or reen color% The relatedhydroymethylfurfuralfrom heoses may ive a

muddy-brown or ray solution+ but this is easily distinuishable from the reen color of pentoses%

5uantitati!e !ersionedit

The test may be performed as a 1uantitative colorimetric test usin a spectrophotometer% &ernell and

Kin have published a procedure for simultaneous determination of pentoses and heoses from

measurements at two wavelenths

6

Larious versions of this test are widely used for a 1uic.chemical determination of


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&rom @i.ipedia+ the free encyclopedia

/his article does not citeanyreferences or sources. 6lease helpimprove this

articlebyadding citations to reliable sources. Bnsourced material may be challenged

and removed. (ecember !"#$)

Molisch's test

Classification +olorimetric method

Analytes +arbohydrates

*olischs test(named after *ustrian botanist Hans Molisch$ is a sensitivechemical testfor thepresence of carbohydrates+based on the dehydration of the carbohydrate by sulfuric acid orhydrochloric acid to produce an aldehyde+ which condenses with two molecules of phenol (usually )-naphthol+thouh other phenols (e%% resorcinol+thymol$ also ive colored products$+ resultin in ared- or purple-colored compound%

Contents

hide

5 rocedure


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https://en.wikipedia.org/wiki/'olisch02Fs$test

/iuret test&rom @i.ipedia+ the free encyclopedia

The characteristic color of a positive biuret test

The #iuret testis a chemical testused for detectin the presence of peptide bonds% /n the presenceof peptides+ a copper(//$ ionformsviolet-coloredcoordination compleesin an al.alinesolution%5!everal variants on the test have been developed+ such as the ,C* test and the Modified Eowrytest%
https://en.wikipedia.org/wiki/Molisch's_testhttps://en.wikipedia.org/wiki/Chemical_testhttps://en.wikipedia.org/wiki/Chemical_testhttps://en.wikipedia.org/wiki/Peptide_bondshttps://en.wikipedia.org/wiki/Copperhttps://en.wikipedia.org/wiki/Ionhttps://en.wikipedia.org/wiki/Violet_(color)https://en.wikipedia.org/wiki/Coordination_complexhttps://en.wikipedia.org/wiki/Coordination_complexhttps://en.wikipedia.org/wiki/Alkalinehttps://en.wikipedia.org/wiki/Biuret_test#cite_note-1https://en.wikipedia.org/wiki/Biuret_test#cite_note-2https://en.wikipedia.org/wiki/Molisch's_testhttps://en.wikipedia.org/wiki/Chemical_testhttps://en.wikipedia.org/wiki/Peptide_bondshttps://en.wikipedia.org/wiki/Copperhttps://en.wikipedia.org/wiki/Ionhttps://en.wikipedia.org/wiki/Violet_(color)https://en.wikipedia.org/wiki/Coordination_complexhttps://en.wikipedia.org/wiki/Alkalinehttps://en.wikipedia.org/wiki/Biuret_test#cite_note-1https://en.wikipedia.org/wiki/Biuret_test#cite_note-2


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The biuret reaction can be used to assess theconcentrationof proteins because peptide bondsoccur with the same fre1uency per amino acid in the peptide% The intensity of the color+ and hencethe absorption at ?#4 nm+ is directly proportional to the protein concentration+ accordin to the ,eer-Eambert law%

2espite its name+ the reaent does not in fact contain biuret((H;-C"-$;H$% The test is so namedbecause it also ives a positive reaction to the peptide-li.e bonds in the biuret molecule%

Contents

5 rocedure

,iuret reaent

6 Hih sensitivity variants of the biuret test

#


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ames

7)stematic EH(6 name

1,2meth)l4,2meth)lphen)ldia@en)l- phen)l-

a@onapthalen2ol

ther names

7udan D8 6.E. 7olvent Ded 248 6.E. 218 ?ipid 6rimson8

il Ded8 il Ded BB8 =at Ded B8 il Ded EI8 7carlet Ded8

7carlet Ded N.=8 7carlet Ded 7charlach8 7carlet D

*denti(ers

6(7 Degistr) Number JJ


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veri+),what is: / -

En+obox re+erences

Sudan /0(C#H4;#"$ is a lysochrome(fat-soluble dye$dia7o dyeused for

thestaininof lipids+trilyceridesandlipoproteinson fro7en paraffinsections% /t has the appearance

of reddish brown crystals with meltin point 5>> DC and maimum absorption at ?4(6?3$ nm%

!udan /L is one of the dyes used for !udan stainin% !imilar dyes include "il >? food safety reulations report%

https://en.wikipedia.org/wiki/7udan$EI

lectrophoresis

&rom @i.ipedia+ the free encyclopedia

For specific types of electrophoresis (for example, the process of administering

medicine, iontophoresis), seeElectrophoresis (disambiguation).
https://en.wikipedia.org/w/index.php?title=Special:ComparePages&rev1=470472512&page2=Sudan+IVhttps://en.wikipedia.org/wiki/Wikipedia:WikiProject_Chemicals/Chembox_validationhttps://en.wikipedia.org/wiki/Wikipedia:Chemical_infobox#Referenceshttps://en.wikipedia.org/wiki/Lysochromehttps://en.wikipedia.org/wiki/Lysochromehttps://en.wikipedia.org/wiki/Diazo_dyehttps://en.wikipedia.org/wiki/Diazo_dyehttps://en.wikipedia.org/wiki/Diazo_dyehttps://en.wikipedia.org/wiki/Staining_(biology)https://en.wikipedia.org/wiki/Staining_(biology)https://en.wikipedia.org/wiki/Lipidshttps://en.wikipedia.org/wiki/Lipidshttps://en.wikipedia.org/wiki/Triglycerideshttps://en.wikipedia.org/wiki/Lipoproteinshttps://en.wikipedia.org/wiki/Lipoproteinshttps://en.wikipedia.org/wiki/Paraffin_waxhttps://en.wikipedia.org/wiki/Sudan_stainhttps://en.wikipedia.org/wiki/Oil_Red_Ohttps://en.wikipedia.org/wiki/Sudan_IIIhttps://en.wikipedia.org/wiki/Sudan_Black_Bhttps://en.wikipedia.org/wiki/Sudan_Black_Bhttps://en.wikipedia.org/wiki/Sudan_Black_Bhttps://en.wikipedia.org/wiki/Propylene_glycolhttps://en.wikipedia.org/wiki/Propylene_glycolhttps://en.wikipedia.org/wiki/Propylene_glycolhttps://en.wikipedia.org/wiki/Propylene_glycolhttp://www.ihcworld.com/_protocols/special_stains/oil_red_o.htmhttps://en.wikipedia.org/wiki/Isopropyl_alcoholhttps://en.wikipedia.org/wiki/Ethanolhttps://en.wikipedia.org/wiki/Ethanolhttps://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttps://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttps://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttps://en.wikipedia.org/wiki/Sudan_Ihttps://en.wikipedia.org/wiki/Sudan_Ihttps://en.wikipedia.org/wiki/Sudan_IIIhttps://en.wikipedia.org/wiki/Carcinogenhttps://en.wikipedia.org/wiki/Carcinogenhttps://en.wikipedia.org/wiki/International_Agency_for_Research_on_Cancerhttps://en.wikipedia.org/wiki/International_Agency_for_Research_on_Cancerhttps://en.wikipedia.org/wiki/Sudan_IV#cite_note-pmid18418879-1https://en.wikipedia.org/wiki/Biebrich_scarlethttps://en.wikipedia.org/wiki/Oilhttps://en.wikipedia.org/wiki/Fathttps://en.wikipedia.org/wiki/Fathttps://en.wikipedia.org/wiki/Waxhttps://en.wikipedia.org/wiki/Grease_(lubricant)https://en.wikipedia.org/wiki/Grease_(lubricant)https://en.wikipedia.org/wiki/Hydrocarbonhttps://en.wikipedia.org/wiki/Hydrocarbonhttps://en.wikipedia.org/wiki/Acryl_grouphttps://en.wikipedia.org/wiki/Acryl_grouphttps://en.wikipedia.org/wiki/Emulsionhttps://en.wikipedia.org/wiki/United_Kingdomhttps://en.wikipedia.org/wiki/Fuel_dyehttps://en.wikipedia.org/wiki/Heating_oilhttps://en.wikipedia.org/wiki/Food_dyehttps://en.wikipedia.org/wiki/Food_dyehttps://en.wikipedia.org/wiki/Carcinogenhttps://en.wikipedia.org/wiki/Sudan_IVhttps://en.wikipedia.org/wiki/Iontophoresishttps://en.wikipedia.org/wiki/Electrophoresis_(disambiguation)https://en.wikipedia.org/wiki/Electrophoresis_(disambiguation)https://en.wikipedia.org/w/index.php?title=Special:ComparePages&rev1=470472512&page2=Sudan+IVhttps://en.wikipedia.org/wiki/Wikipedia:WikiProject_Chemicals/Chembox_validationhttps://en.wikipedia.org/wiki/Wikipedia:Chemical_infobox#Referenceshttps://en.wikipedia.org/wiki/Lysochromehttps://en.wikipedia.org/wiki/Diazo_dyehttps://en.wikipedia.org/wiki/Staining_(biology)https://en.wikipedia.org/wiki/Lipidshttps://en.wikipedia.org/wiki/Triglycerideshttps://en.wikipedia.org/wiki/Lipoproteinshttps://en.wikipedia.org/wiki/Paraffin_waxhttps://en.wikipedia.org/wiki/Sudan_stainhttps://en.wikipedia.org/wiki/Oil_Red_Ohttps://en.wikipedia.org/wiki/Sudan_IIIhttps://en.wikipedia.org/wiki/Sudan_Black_Bhttps://en.wikipedia.org/wiki/Propylene_glycolhttp://www.ihcworld.com/_protocols/special_stains/oil_red_o.htmhttps://en.wikipedia.org/wiki/Isopropyl_alcoholhttps://en.wikipedia.org/wiki/Ethanolhttps://en.wikipedia.org/wiki/Wikipedia:Citation_neededhttps://en.wikipedia.org/wiki/Sudan_Ihttps://en.wikipedia.org/wiki/Sudan_IIIhttps://en.wikipedia.org/wiki/Carcinogenhttps://en.wikipedia.org/wiki/International_Agency_for_Research_on_Cancerhttps://en.wikipedia.org/wiki/Sudan_IV#cite_note-pmid18418879-1https://en.wikipedia.org/wiki/Biebrich_scarlethttps://en.wikipedia.org/wiki/Oilhttps://en.wikipedia.org/wiki/Fathttps://en.wikipedia.org/wiki/Waxhttps://en.wikipedia.org/wiki/Grease_(lubricant)https://en.wikipedia.org/wiki/Hydrocarbonhttps://en.wikipedia.org/wiki/Acryl_grouphttps://en.wikipedia.org/wiki/Emulsionhttps://en.wikipedia.org/wiki/United_Kingdomhttps://en.wikipedia.org/wiki/Fuel_dyehttps://en.wikipedia.org/wiki/Heating_oilhttps://en.wikipedia.org/wiki/Food_dyehttps://en.wikipedia.org/wiki/Food_dyehttps://en.wikipedia.org/wiki/Carcinogenhttps://en.wikipedia.org/wiki/Sudan_IVhttps://en.wikipedia.org/wiki/Iontophoresishttps://en.wikipedia.org/wiki/Electrophoresis_(disambiguation)


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/llustration of electrophoresis

/llustration of electrophoresis retardation

lectrophoresisis the motion ofdispersed particlesrelative to a fluid under the influence of a

spatially uniform electric field%56#?Thiselectro.inetic phenomenonwas observed for the first time

in 5943 by &erdinand &rederic


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used for plasmid analysis+ which develops our understandin of bacteria becomin resistant to

antibiotics%

https://en.wikipedia.org/wiki/lectrophoresis

2el electrophoresis&rom @i.ipedia+ the free encyclopedia

-el electrophoresis

Oel electrophoresis apparatus Q (n agarose gel is placed

in this buCer#lled box and an electrical #eld is applied

via the power suppl) to the rear. %he negative terminal

is at the +ar end ,black wire-8 so *N( migrates toward

the anode ,red wire-.

Classi(cation lectrophoresis

ther techni/)es

Related 6apillar) electrophoresis

7*7(O

%wodimensional gel electrophoresis

%emperature gradient gel
https://en.wikipedia.org/wiki/Electrophoresishttps://en.wikipedia.org/wiki/Electrophoresishttps://en.wikipedia.org/wiki/Capillary_electrophoresishttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttps://en.wikipedia.org/wiki/Temperature_gradient_gel_electrophoresishttps://en.wikipedia.org/wiki/Electrophoresishttps://en.wikipedia.org/wiki/Electrophoresishttps://en.wikipedia.org/wiki/Capillary_electrophoresishttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresishttps://en.wikipedia.org/wiki/Temperature_gradient_gel_electrophoresis


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electrophoresis

2iital imae of 6 plasmid restriction diests run on a 5I w0v aarose el+ 6 volt0cm+ stained with ethidium

bromide% The 2;* si7e mar.er is a commercial 5 .bp ladder% The position of the wells and direction of 2;*

miration is noted%

3el electrophoresisis a method for separation and analysis of macromolecules

(2;*+


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retardin the passae of moleculesJ els can also simply serve to maintain the finished separation+

so that a post electrophoresis stain can be applied%62;* Gel electrophoresis is usually performed

for analytical purposes+ often after amplification of 2;* via C


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/n simple terms+ electrophoresisis a process which enables the sortin of molecules based on si7e%

Fsin an electric field+ molecules (such as 2;*$ can be made to move throuh a el made

of aaror polyacrylamide% The electric field consists of a neative chare at one end which pushes

the molecules throuh the el+ and a positive chare at the other end that pulls the molecules

throuh the el% The molecules bein sorted are dispensed into a well in the el material% The el is

placed in an electrophoresis chamber+ which is then connected to a power source% @hen the electriccurrent is applied+ the larer molecules move more slowly throuh the el while the smaller

molecules move faster% The different si7ed molecules form distinct bands on the el% citation needed

The term NelN in this instance refers to the matri used to contain+ then separate the taret

molecules% /n most cases+ the el is acrosslin.ed polymerwhose composition and porosity is

chosen based on the specific weiht and composition of the taret to be analy7ed% @hen

separatin proteinsor small nucleic acids(2;*+


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on pH+ but runnin for too lon can ehaust the bufferin capacity of the solution% &urther+ different

preparations of enetic material may not mirate consistently with each other+ for morpholoical or

other reasons%

$ypes of geledit

The types of el most typically used are aarose and polyacrylamide els% ach type of el is well-

suited to different types and si7es of analyte% olyacrylamide els are usually used for proteins+ and

have very hih resolvin power for small framents of 2;* (?-?44 bp$% *arose els on the other

hand have lower resolvin power for 2;* but have reater rane of separation+ and are therefore

used for 2;* framents of usually ?4-4+444 bp in si7e+ but resolution of over Mb is possible

withpulsed field el electrophoresis(&G$%?olyacrylamide els are run in a vertical confiuration

while aarose els are typically run hori7ontally in a submarine mode% They also differ in their

castin methodoloy+ as aarose sets thermally+ while polyacrylamide forms in a chemical

polymeri7ation reaction%

&garoseeditMain article:Agarose gel electrophoresis

*arose els are made from the natural polysaccharidepolymersetracted fromseaweed% *arose

els are easily cast and handled compared to other matrices+ because the el settin is a physical

rather than chemical chane% !amples are also easily recovered% *fter the eperiment is finished+

the resultin el can be stored in a plastic ba in a refrierator%

*arose els do not have a uniform pore si7e+ but are optimal for electrophoresis of proteins that are

larer than 44 .2a%*arose el electrophoresis can also be used for the separation of 2;*

framents ranin from ?4 base pairto several meabases (millions of bases$+ the larest of which

re1uire speciali7ed apparatus% The distance between 2;* bands of different lenths is influenced by

the percent aarose in the el+ with hiher percentaes re1uirin loner run times+ sometimes days%

/nstead hih percentae aarose els should be run with apulsed field electrophoresis(&$+

or field inversion electrophoresis%

NMost aarose els are made with between 4%3I (ood separation or resolution of lare ?=54.b

2;* framents$ and I (ood resolution for small 4%=5.b framents$ aarose dissolved in

electrophoresis buffer% Fp to 6I can be used for separatin very tiny framents but a vertical

polyacrylamide el is more appropriate in this case% Eow percentae els are very wea. and may

brea. when you try to lift them% Hih percentae els are often brittle and do not set evenly% 5I els

are common for many applications%N3

PolyacrylamideeditMain article:olyacrylamide gel electrophoresis

olyacrylamide el electrophoresis (*G$ is used for separatin proteins ranin in si7e from ? to

+444 .2a due to the uniform pore si7e provided by the polyacrylamide el% ore si7e is controlled

by modulatin the concentrations of acrylamide and bis-acrylamide powder used in creatin a el%
https://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=2https://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-5https://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=3https://en.wikipedia.org/wiki/Agarose_gel_electrophoresishttps://en.wikipedia.org/wiki/Agarose_gel_electrophoresishttps://en.wikipedia.org/wiki/Polysaccharidehttps://en.wikipedia.org/wiki/Polymershttps://en.wikipedia.org/wiki/Seaweedhttps://en.wikipedia.org/wiki/Seaweedhttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-6https://en.wikipedia.org/wiki/Base_pairhttps://en.wikipedia.org/wiki/Base_pairhttps://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/w/index.php?title=Field_inversion_electrophoresis&action=edit&redlink=1https://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-7https://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=4https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresishttps://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresishttps://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=2https://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-5https://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=3https://en.wikipedia.org/wiki/Agarose_gel_electrophoresishttps://en.wikipedia.org/wiki/Polysaccharidehttps://en.wikipedia.org/wiki/Polymershttps://en.wikipedia.org/wiki/Seaweedhttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-6https://en.wikipedia.org/wiki/Base_pairhttps://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresishttps://en.wikipedia.org/w/index.php?title=Field_inversion_electrophoresis&action=edit&redlink=1https://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-7https://en.wikipedia.org/w/index.php?title=Gel_electrophoresis&action=edit&section=4https://en.wikipedia.org/wiki/Polyacrylamide_gel_electrophoresis


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Care must be used when creatin this type of el+ as acrylamide is a potent neurotoin in its li1uid

and powdered forms%

Traditional2;* se1uencintechni1ues such asMaam-Gilbertor!anermethods used

polyacrylamide els to separate 2;* framents differin by a sinle base-pair in lenth so the

se1uence could be read% Most modern 2;* separation methods now use aarose els+ ecept forparticularly small 2;* framents% /t is currently most often used in the field of immunoloyand

protein analysis+ often used to separate different proteins orisoformsof the same protein into

separate bands% These can be transferred onto anitrocelluloseor L2&membrane to be probed

with antibodies and correspondin mar.ers+ such as in a western blot%

Typically resolvin elsare made in I+ 9I+ 54I+ 5I or 5?I% !tac.in el (?I$ is poured on top

of the resolvin el and a el comb (which forms the wells and defines the lanes where proteins+

sample buffer and ladders will be placed$ is inserted% The percentae chosen depends on the si7e of

the protein that one wishes to identify or probe in the sample% The smaller the .nown weiht+ the

hiher the percentae that should be used% Chanes on the buffer system of the el can help to

further resolve proteins of very small si7es%9

Starchedit

artially hydrolysedpotato starch ma.es for another non-toic medium for protein electrophoresis%

The els are slihtly more opa1ue than acrylamide or aarose% ;on-denatured proteins can be

separated accordin to chare and si7e% They are visualised usin ;apthal ,lac. or *mido ,lac.

stainin% Typical starch el concentrations are ?I to 54I%>5455

2el conditionsedit

Denaturingedit

TTG profiles representin the bifidobacterial diversity of fecal samples from two healthy volunteers (* and ,$

before and after *MC ("ral *moicillin-Clavulanic *cid$ treatment

2enaturinels are run under conditions that disrupt the natural structure of the analyte+ causin it

to unfold into a linear chain% Thus+ the mobility of each macromoleculedepends only on its linear

lenth and its mass-to-chare ratio% Thus+ the secondary+ tertiary+ and 1uaternary levels

of biomolecular structureare disrupted+ leavin only the primary structure to be analy7ed%

;ucleic acids are often denatured by includin ureain the buffer+ while proteins are denatured

usin sodium dodecyl sulfate+ usually as part of the!2!-*Gprocess% &or full denaturation ofproteins+ it is also necessary to reduce the covalent disulfide bondsthat stabili7e

theirtertiaryand 1uaternary structure+ a method called reducin *G%


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mobility%Frea+2M!"and lyoalare the most often used denaturin aents to disrupt


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Fnli.e denaturin methods+ native el electrophoresis does not use a chareddenaturinaent%

The molecules bein separated (usuallyproteinsor nucleic acids$ therefore differ not only

in molecular massand intrinsic chare+ but also the cross-sectional area+ and thus eperience

different electrophoretic forces dependent on the shape of the overall structure% &or proteins+ since

they remain in the native state they may be visualised not only by eneral protein stainin reaents

but also by specific en7yme-lin.ed stainin%

;ativeel electrophoresis is typically used inproteomicsand metallomics% However+ native *G is

also used to scan enes (2;*$ for un.nown mutations as in !inle-strand conformation

polymorphism%

/uffersedit

,uffers in el electrophoresis are used to provide ions that carry a current and to maintain the pH at

a relatively constant value% There are a number of buffers used for electrophoresis% The most

common bein+ for nucleic acids Tris0*cetate02T*(T*$+ Tris0,orate02T*(T,$% Many other

buffers have been proposed+ e%% lithium borate+ which is almost never used+ based on ubmed

citations (E,$+ iso electric histidine+ pK matched oods buffers+ etc%J in most cases the purported

rationale is lower current (less heat$ and or matched ion mobilities+ which leads to loner buffer life%

,orate is problematicJ ,orate can polymeri7e+ and0or interact with cis diols such as those found in


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eample in a2;* se1uencinel+ an autoradioramcan be recorded of the el%hotoraphscan

be ta.en of els+ often usin a Gel 2ocsystem%

Downstream processingedit

*fter separation+ an additional separation method may then be used+ such as isoelectric

focusinor !2!-*G% The el will then be physically cut+ and the protein complees etracted

from each portion separately% ach etract may then be analysed+ such as by peptide mass

finerprintinor de novo peptide se1uencinafter in-el diestion% This can provide a reat deal of

information about the identities of the proteins in a comple%

Applicationsedit

"verview of Gel lectrophoresis%

stimation of the si7e of 2;* molecules followin restriction en7yme diestion+ e%%

in restriction mappinof cloned 2;*%

*nalysis of C


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*n aarose el of a C


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SDS-P&3autoradiography= The indicated proteins are present in different concentrations in the two

samples%

roteins+ unli.e nucleic acids+ can have varyin chares and comple shapes+ therefore they may

not mirate into the polyacrylamide el at similar rates+ or at all+ when placin a neative to positive

M& on the sample% roteins therefore+ are usually denaturedin the presence of a deterentsuch

as sodium dodecyl sulfate(!2!$ that coats the proteins with a neative chare%6Generally+ the

amount of !2! bound is relative to the si7e of the protein (usually 5%# !2! per ram of protein$+ so

that the resultin denatured proteins have an overall neative chare+ and all the proteins have a

similar chare to mass ratio% !ince denatured proteins act li.e lon rods instead of havin a comple

tertiary shape+ the rate at which the resultin !2! coated proteins mirate in the el is relative only

to its si7e and not its chare or shape%6

roteinsare usually analy7ed by sodium dodecyl sulfate polyacrylamide el electrophoresis (!2!-

*G$+ by native el electrophoresis+ by 1uantitative preparative native continuous polyacrylamide

el electrophoresis (Q;C-*G$+ or by-2 electrophoresis%

Characteri7ation throuh liand interaction may be performed byelectroblottinor by affinity

electrophoresisin aarose or bycapillary electrophoresisas for estimation of bindin constantsand

determination of structural features li.e lycancontent throuh lectinbindin%

https://en.wikipedia.org/wiki/Oel$electrophoresis

Countercurrent distri#ution

Chemistry &D

0/ (/ST!+

D/T
https://en.wikipedia.org/wiki/Autoradiographyhttps://en.wikipedia.org/wiki/Autoradiographyhttps://en.wikipedia.org/wiki/Autoradiographyhttps://en.wikipedia.org/wiki/Proteinhttps://en.wikipedia.org/wiki/Denaturation_(biochemistry)https://en.wikipedia.org/wiki/Denaturation_(biochemistry)https://en.wikipedia.org/wiki/Detergenthttps://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-Stryer-3https://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-Stryer-3https://en.wikipedia.org/wiki/Proteinhttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/Native_Gel_Electrophoresishttps://en.wikipedia.org/wiki/QPNC-PAGEhttps://en.wikipedia.org/wiki/2-D_electrophoresishttps://en.wikipedia.org/wiki/2-D_electrophoresishttps://en.wikipedia.org/wiki/Electroblottinghttps://en.wikipedia.org/wiki/Electroblottinghttps://en.wikipedia.org/wiki/Affinity_electrophoresishttps://en.wikipedia.org/wiki/Affinity_electrophoresishttps://en.wikipedia.org/wiki/Affinity_electrophoresishttps://en.wikipedia.org/wiki/Capillary_electrophoresishttps://en.wikipedia.org/wiki/Capillary_electrophoresishttps://en.wikipedia.org/wiki/Binding_constanthttps://en.wikipedia.org/wiki/Binding_constanthttps://en.wikipedia.org/wiki/Glycanhttps://en.wikipedia.org/wiki/Glycanhttps://en.wikipedia.org/wiki/Lectinhttps://en.wikipedia.org/wiki/Gel_electrophoresishttp://www.britannica.com/science/countercurrent-distribution/article-historyhttp://www.britannica.com/science/countercurrent-distributionhttps://en.wikipedia.org/wiki/Autoradiographyhttps://en.wikipedia.org/wiki/Proteinhttps://en.wikipedia.org/wiki/Denaturation_(biochemistry)https://en.wikipedia.org/wiki/Detergenthttps://en.wikipedia.org/wiki/Sodium_dodecyl_sulfatehttps://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-Stryer-3https://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-Stryer-3https://en.wikipedia.org/wiki/Proteinhttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/SDS-PAGEhttps://en.wikipedia.org/wiki/Native_Gel_Electrophoresishttps://en.wikipedia.org/wiki/QPNC-PAGEhttps://en.wikipedia.org/wiki/2-D_electrophoresishttps://en.wikipedia.org/wiki/Electroblottinghttps://en.wikipedia.org/wiki/Affinity_electrophoresishttps://en.wikipedia.org/wiki/Affinity_electrophoresishttps://en.wikipedia.org/wiki/Capillary_electrophoresishttps://en.wikipedia.org/wiki/Binding_constanthttps://en.wikipedia.org/wiki/Glycanhttps://en.wikipedia.org/wiki/Lectinhttps://en.wikipedia.org/wiki/Gel_electrophoresishttp://www.britannica.com/science/countercurrent-distribution/article-historyhttp://www.britannica.com/science/countercurrent-distribution


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FD"&C6

countercurrent distri#ution7 in chemistry+ a multistaesolvent-etractionprocess+

one of many separation methods that can be employed in chemical analysis%

!ubstances are separated by this method on the basis of their different solubilities in

two immiscible li1uids% These two li1uids+ flowin in opposite directions+ are brouht into

contact+ mied+ and allowed to separate% The upper layer is transferred off in one

direction and the lower in anotherJ this cycle of operations may be repeated as many

times as necessary to effect the desired separation%

* sample of a substance in contact with two solvents that do not dissolve in one another

see.s an e1uilibrium condition in which it is distributed between themJ the ratio of the

concentrations in the two solvents+ called the distribution coefficient+ is characteristic of

the compound and of the solvent pair% Compounds that have dissimilar molecularstructures usually have widely different distribution coefficients+ and mitures of such

compounds can be separated satisfactorily by one or a few transfers between a suitable

solvent pair in simple e1uipment% Closely similar substances+ however+ such as

proteins+ have very similar distribution coefficients+ and hundreds of transfers may be

re1uired to produce a complete separation%

The principle of countercurrent distribution is similar to that of chromatoraphyJ both

procedures are used for analysis and purification of mitures of similar compounds%

http://www.britannica.com/science/countercurrentdistribution

Co)nterc)rrent 0traction " Crai2 Apparat)s.

3heory

( method o+ multiple liGuidliGuid extractions is countercurrent extraction8 which permits theseparation o+ substances with diCerent distribution coeRcients ,ratios-. ( clever designknown as 6raig apparatus is used +or this purpose ,?)man 6. 6raig8 194


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?)man 6. 6raig8 1919F4 ,Biograph)-

6raig apparatus consists o+ a series o+ glass tubes ,r: 8 18 2..- that are designed andarranged such that the lighter liGuid phase is trans+erred +rom one tube to the next. %heliGuidliGuid extractions are taking place simultaneousl) in all tubes o+ the apparatus whichis usuall) driven electromechanicall). En the +ollowing animated picture o+ a single glass tubethe t)pical Sextraction/trans+erS c)cle is shown.

%he lower ,heavier- phase o+ the twophase solvent s)stem ,e.g. water8 blue la)er in thepicture- is the Sstationar) phaseS8 whereas the upper ,lighter- phase ,e.g. hexane8 red la)erin the picture- is the Smobile phaseS.

En the beginning8 tube T contains the mixture o+ substances to be separated in the heaviersolvent and all the other tubes contain eGual volumes o+ the same solvent. %he lightersolvent is added to tube T8 extraction ,eGuilibration- takes place and the phases areallowed to separate. %he upper phase o+ tube T is then trans+erred to tube T1 and +reshsolvent is added to tube T8 and the phases are eGuilibrated again. %he upper la)ers o+tubes T and T1 are simultaneousl) trans+erred to tubes T1 and T2 respectivel). %his c)cleis repeated to carr) on the process through the other tubes o+ the apparatus. bviousl)8substances with higher distribution ratio move +aster than those with a lower distributionratio.

Et is interesting to examine the distribution o+ a substance ( in each tube a+ter a givennumber o+ eGuilibration/trans+er c)cles.

7upposing that the volumes o+ each solvent are eGual ,I-8 and let > represent the weight o+( in the sample8 p and G represent the +raction o+ ( with distribution ratio o+ * in the upper,organic solvent8 o- and lower ,water8 w- phase8 then it is

7ince pUG ! 18 we have

%he +ractions o+ solute in successive tubes a+ter each extraction step are shown in the+ollowing #gure:
http://www.jbc.org/cgi/content/full/280/7/e4http://www.jbc.org/cgi/content/full/280/7/e4


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>e observe that a+ter n trans+ers/eGuilibration c)cles8 and since the ratio *!p/G must bemaintained +or each tube a+ter the eGuilibration step8 the total +raction o+ ( in each tubecorresponds to the terms o+ the binomial expansion ,pUG-n. %here+ore the total +raction o+solute in tube r a+ter n trans+ers is given b) ,remember that V!1-.

B) combining with the previous expressions o+ p and G we #nall) obtain

%he greater the diCerence o+ the distribution ratio o+ various substances8 the better theseparation between each other. ( much larger number o+ tubes is reGuired to separatemixtures o+ substances with almost similar distribution ratios.

%he 6raig apparatus is onl) rarel) used because modern chromatographic techniGues are b)+ar more eRcient and convenient. 5owever countercurrent extraction is educationall) ver)use+ul since it introduces the student to the +undamental concept o+ eGuilibration betweenmobile and static phases. ach tube where a complete eGuilibration takes place correspondsto one theoretical plate o+ the chromatographic column. 6raig apparatuses with more thanabout 1 tubes are ver) diRcult are ver) diRcult to construct and operate8 and the) mustbe compared with modern chromatographic columns that ma) exhibit eRciencies o+ tens o+thousands theoretical plates.


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( manuall) operated 6raig apparatus consisting o+ 2 tubes ,+rom:www.theliGuidphase.org8W histor)-.

Applet

%his applet demonstrates the principles o+ countercurrent extraction. ( mixture o+ twosubstances: ( ,with green color- and B ,with red color- is going to be separated using a 6raigapparatus consisting o+ 2 ,8 18 .. 19- eGuilibration tubes. %he user must enter the values o+distribution ratios +or the two substances in the corresponding edit boxes.

B) clicking the button 7%(D% the experiment starts with tube T containing the mixture inthe lower phase8 pure ,lightersolvent in the upper phase and the other tubes ,T1T19-containing pure ,heaviersolvent-.

B) clicking the button xtract an eGuilibration takes place and the two substances aredistributed between the lower and the upper phase according to their distribution ratio.%hen8 b) clicking the button %rans+er the upper phase o+ tube T is trans+er to tube T1 and+resh upper phase to tube T. %he c)cle can be repeated as man) times we wish.

%he eCectiveness o+ the separation ,+or the given values o+ distribution ratios- can beobserved b) both the bargram indicating the +raction o+ each substance in each tube andthe intensit) o+ the colors in the upper and lower phases in each tube.

(lso8 the eluted +raction ,percent- o+ each substance +rom the last tube is shown.

http://www.chem.uoa.gr/applets/(pplet6raig/(ppl$6raig2.html

Centrifuation and centrifues

Centrifugation is a separation process which uses the action of centrifugal force to promote

accelerated settling of particles in a solid-liquid mixture. Two distinct major phases are formed in

the vessel during centrifugation :

Thesediment

Usually does not have a uniform structure.

Find below an example of a sediment deposit :
http://www.theliquidphase.org/http://www.theliquidphase.org/http://www.theliquidphase.org/http://www.chem.uoa.gr/applets/AppletCraig/Appl_Craig2.htmlhttp://www.theliquidphase.org/http://www.chem.uoa.gr/applets/AppletCraig/Appl_Craig2.html


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Thecentrifugateorcentratewhich is the supernatant liquid.

Often clear though sometimes cloudy, due to the presence of very finecolloidal particlesthat are

not readily settled. However it may also contain several phases if the mixtures interstitial liquid

contains element with different densities, such as oils for example.

*E/C*T/"; /; @*T< T


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rotation. This can either replace normal gravity in the sedimentation of suspension or provide the

driving force in the filtration through a filter medium of some kind.

The most common application is separation of solid substances from high concentrated

suspensions. Used in this way for the treatment of sewage sludge it enables the dewatering with

the production of more or less consistent sediment depending on the nature of the sludge to be

treated, and the accelerated thickening of low concentration sludge.

Principle

The separation is similar in principle to that achieved in a gravity separation process. The driving

force is higher because is resulting from the rotation of the liquid: in the case of sedimentation,

where the driving force is resulting from the difference in density between the solids particles and

the liquid, the separation is achieved with a force from 1000 to 20000 times that of gravity.

Types

Most centrifuges rotate thanks to some kind of motor drive. The types of centrifuge used for

sedimentation include:

hydrocyclone

tubular bowl

chamber bowl

imperforate basket

disk stack separator

decanter

Sedimenting centrifuges were invented for liquid solid separation and not for handling solids. It

soon became apparent that these machines had wider applications, which would involve the

presence of solid impurities, leading to use for separating solids from liquids.

http://www.lenntech.com/librar)/clari#cation/clari#cation/centri+ugation.htm

Centrif)2ation 3heory

Centrifugationis a process used to separate or concentratematerials suspended in a liGuid medium. %he theoretical basis o+ thistechniGue is the eCect o+ gravit) on particles ,including

macromolecules- in suspension. %wo particles o+ diCerent masseswill settle in a tube at diCerent rates in response to gravit).6entri+ugal +orce ,measured as xg8 gravit)- is used to increase thissettling rate in an instrument called a centri+uge. %wo commonexamples o+ the use o+ centri+ugal +orce are: ,1- >hen )ou do theSaround the worldS trick with a )o)o8 it is centri+ugal +orce thatmakes the )o)o bod) sta) at the end o+ the string as )ou rotate it
http://www.lenntech.com/Centrifugation.htm#Hydrocyclonehttp://www.lenntech.com/Centrifugation.htm#Tubular_bowl_centrifugehttp://www.lenntech.com/Centrifugation.htm#Chamber_bowl_centrifugehttp://www.lenntech.com/Centrifugation.htm#Imperforate_basket_centrifugehttp://www.lenntech.com/Centrifugation.htm#Disk_stack_separatorhttp://www.lenntech.com/Centrifugation.htm#Decanterhttp://www.lenntech.com/library/clarification/clarification/centrifugation.htmhttp://www.lenntech.com/Centrifugation.htm#Hydrocyclonehttp://www.lenntech.com/Centrifugation.htm#Tubular_bowl_centrifugehttp://www.lenntech.com/Centrifugation.htm#Chamber_bowl_centrifugehttp://www.lenntech.com/Centrifugation.htm#Imperforate_basket_centrifugehttp://www.lenntech.com/Centrifugation.htm#Disk_stack_separatorhttp://www.lenntech.com/Centrifugation.htm#Decanterhttp://www.lenntech.com/library/clarification/clarification/centrifugation.htm


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and ,2- >hen )ou wash clothes in a washing machine8 it iscentri+ugal +orce generated in the SspinS c)cle that +orces water outo+ the +abric to +acilitate +aster dr)ing.

6entri+uges are devices used in a variet) o+ scienti#c and technical

applications which spin carrier vessels ,centri+uge tubes- at highrotation speeds and ver) high centri+ugal +orce. %he centri+ugal +orce,expressed as T gravitiesor8 T xg- generated is proportional to therotation rate o+ the rotor ,in rpm- and the distance between the rotorcenter and the centri+uge tube. %here+ore8 a given centri+uge ma)use multiple rotor si@es to give Xexibilt) in choosing centri+ugationconditions. ach centri+uge has a special graph8 a nomograph8 or atable which relates rotation rate ,rpm- to centri+ugal +orce ,xg- +oreach si@e o+ rotor it accepts.

%)picall)8 the material to be SspunS is placed in a centri+uge tubewhich is then placed in a rotor. %he rotor is generall) a dense metalwhich dissipates heat Guickl)8 and is o+ suRcient mass that itgenerates momentum8 i.e.8 once its spinning it reGuires little energ)to keep it going. 6entri+uges generall) work under vacuum and arere+rigerated to reduce heating caused b) +rictional +orces as therotor spins. Dotors are usuall) stored in re+rigeration units to keepthem at or near the operating temperature.

6entri+uges come in all shapes and si@es8 and the rotors var)8there+ore8 the universal and trans+erable unit o+ centri+ugation iscentri+ugal +orce in gravities ,xg-. *iCerent makes o+ centri+uges usediCerent rotors and each model comes with a table or a graph thatrelates centri+ugal +orce to rotational speed ,rpm- +or each rotor ,orswing buckets- it can use. En lab writeups )ou should (?>(Y7 reportthe centri+ugal +orce used ,Tgravities- and duration o+ time at that+orce because centri+ugal +orce is the onl) trans+erable unit betweendiCerent centri+uges.

%op o+ page

Di4erential Centrif)2ation

( commonl) used techniGue +or cell +ractionation8 called dierentialcentrifugation8 is used to separate particles +rom a liGuid medium orto separate particles o+ diCerent masses into separate +ractions o+
http://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html#topofpagehttp://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html#topofpage


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the supernatant. >e will use this techniGue in a several wa)s in thiscourse.

1. En the Bio 242 (m)lase lab we will use centri+ugation to pellet thecellular debris and excess starch during the en@)me extract

preparation. %he en@)me8 which is soluble8 will remain in thesupernatant. *uring the actual experiment8 we will usecentri+ugation to separate the en@)me ,soluble- +rom its substrate,insoluble am)losea@ure- to stop the reaction.

2. En the molecular labs we will use centri+ugation to promote achemical reaction b) +orcing small Guantities o+ reactants together inthe bottom o+ microcentri+uge tubes. >e will also use centri+ugationto prepare bacterial cells +or trans+ormation b) alternatel) pelletingthem and then resuspending them with diCerent chemical solutions.


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protein extraction +or diCerent species o+ organisms and diCerent cell and

tissue t)pes reGuire diCerent buCer +ormulations. %his article brieX)

describes available reagents +or whole cell l)sis and protein extraction. =or a

discussion o+ cell +ractionation and organelle isolation8 see the link in the

sidebar.

5earn more

"verview of cell lysis

Traditional cell lysis methods

2eterents for cell lysis

Cell lysis )sin2 deter2ents

2eterent cell lysis is a milder and easier alternative to physical disruption of cell membranes+ althouh it

is often used in conunction with homoeni7ation and mechanical rindin% 2eterents brea. the lipid

barrier surroundin cells by solubili7in proteins and disruptin lipid:lipid+ protein:protein and protein:lipid

interactions% 2eterents+ li.e lipids+ self associate and bind to hydrophobic surfaces% They are comprised

of a polar hydrophilic head roup and a nonpolar hydrophobic tail and are cateori7ed by the nature of thehead roup as either ionic (cationic or anionic$+ nonionic or 7witterionic% Their behavior depends on the

properties of the head roup and tail%

Fnfortunately+ there is no standard protocol available for selectin a deterent to use for membrane lysis%

/n eneral+ nonionic and 7witterionic deterents are milder and less denaturin than ionic deterents and

are used to solubili7e membrane proteins where it is critical to maintain protein function and0or retain

native protein:protein interactions for en7yme assays or immunoassays% CH*!+ a 7witterionic deterent+

and the Triton-R series of nonionic deterents are commonly used for these purposes% /n contrast+ ionic

deterents are stron solubili7in aents and tend to denature proteins+ thereby destroyin protein activity

and function%

The choice of deterent for cell lysis also depends on sample type% *nimal cells+ bacteria and yeast all

have differin re1uirements for optimal lysis due to the presence or absence of a cell wall% ,ecause of the

dense and comple nature of animal tissues+ they re1uire both deterent and mechanical lysis% /n addition

to the choice of deterent+ other important considerations for optimal cell lysis include the buffer+ pH+ salt

concentration and temperature% Consideration should be iven to the compatibility of the chosen
https://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-cell-lysis-and-protein-extraction.htmlhttps://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.htmlhttps://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/detergents-cell-lysis-protein-extraction.htmlhttps://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-cell-lysis-and-protein-extraction.htmlhttps://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/traditional-methods-cell-lysis.htmlhttps://www.lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/detergents-cell-lysis-protein-extraction.html


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deterent with downstream applications% /f the deterent used for lysis must be removed+ then a

dialy7able deterent should be selected%

https://www.li+etechnologies.com/ph/en/home/li+escience/proteinbiolog)/protein

biolog)learningcenter/proteinbiolog)resourcelibrar)/pierceproteinmethods/cell

l)sissolutions.html

http://www.sciencedirect.com/science/article/pii/72F


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Ded blood cells8 +ound in man) larger animals8 react to higher and lower concentrations o+ solution in blood.

Related Articles Biolog) 6omponents o+ an (nimal 6ell

>hat 6ell 7tructure 6auses hotos)nthesis

>hat >ould 5appen i+ a 6r)stal o+ a 7olute >as (dded to an Hnsaturated 7olution >hat =orms (cross the 6enter o+ a 6ell Near the nd o+ %elophase

Both plants and animals have cells8 and one o+ the main diCerences between them is that

plant cells have a cell wall. %his helps the cells retain their shape even i+ their environment

changes considerabl). (nimal cells are more Xexible8 and without the cell wall8 the) can

react more adversel) to changes in their environment8 such as the concentration o+ a

solution around them.

lasma 'embrane

(ll cells are surrounded b) a plasma membrane8 which is a double la)er o+ phospholipidsthat regulate the transport o+ materials into and out o+ the cell. En a plant8 the cell

membrane is next to the cell wall8 which helps give it structure. %he plasma membrane o+ a

cell is said to be diCerentiall) permeable because it allows certain substances8 such as

water8 to Xow +reel) through it8 but not others8 such as ions and large molecules.

*iCusion and smosis
http://classroom.synonym.com/biology-components-animal-cell-23748.htmlhttp://classroom.synonym.com/cell-structure-causes-photosynthesis-20882.htmlhttp://classroom.synonym.com/would-happen-crystal-solute-added-unsaturated-solution-12007.htmlhttp://classroom.synonym.com/forms-across-center-cell-near-end-telophase-8162.htmlhttp://classroom.synonym.com/biology-components-animal-cell-23748.htmlhttp://classroom.synonym.com/cell-structure-causes-photosynthesis-20882.htmlhttp://classroom.synonym.com/would-happen-crystal-solute-added-unsaturated-solution-12007.htmlhttp://classroom.synonym.com/forms-across-center-cell-near-end-telophase-8162.html


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( solution contains the solvent8 the liGuid8 such as water and the solute8 the solids dissolved

into the solvent. En a solution8 particles in an area o+ high concentration will naturall) move

to an area o+ low concentration. %his is known as diCusion. >hen diCusion happens across a

diCerentiall) permeable membrane8 it is known as osmosis. %his is important to cells

because i+ the solute cannot move across the diCerentiall) permeable membrane8 water will

+reel) move into or out o+ a cell to balance out the concentration.

Esotonic 7olution

E+ a cell is in a solution that has the same concentration outside the cell as it is inside the

cell8 it is said to be isotonic. %his is the ideal situation +or a cell8 and the Xow o+ water into

the cell eGuals the Xow o+ water out o+ the cell. 5owever8 cells o+ten encounter environments

that have diCerent concentrations o+ solution8 such as organisms +ound in +resh water ,low

concentration o+ solute- or ocean water ,high concentration o+ solute-.

5)potonic 7olution

E+ the solution surrounding a cell is less concentrated than that inside the cell8 it is said to be

h)potonic. En a h)potonic solution8 water will move +rom outside to the inside o+ the cell

across its membrane. %his process will continue until the solutions reach eGual

concentration8 the cell ruptures +rom internal pressure or organelles inside the cell exert

energ) to pump water out o+ the cell.

5)pertonic 7olution

E+ a cell is in a solution that has a higher concentration than what is inside the cell8 it is said

to be h)pertonic. >hen this happens8 water will Xow out o+ the cell and into the solution.

%his will continue until the concentrations are balanced8 the cell deh)drates and collapses orthe organelles in the cell exert energ) to pump water back into the cell.

(nimal 6ells vs. lant 6ells

>hen animal cells are placed in a h)potonic solution8 the) will take in water due to osmosis.

E+ the solution in which the) are placed is a low enough concentration8 such as distilled

water8 the intake o+ water will make the cells swell up and eventuall) burst. %his can8 o+

course8 be a ver) dangerous result +or cells placed in a solution o+ low concentration. lant

cells do not rupture because the rigid cell walls hold them together.

http://classroom.s)non)m.com/happensanimalcellh)potonicsolution2F.html

/s distilled water hypertonic orhypotonicS
http://classroom.synonym.com/happens-animal-cell-hypotonic-solution-2607.htmlhttp://classroom.synonym.com/happens-animal-cell-hypotonic-solution-2607.html


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&8

9'/C6 &4S

ith regard to osmosis7 distilled water will always #e hypotonic compared to an

aueous solution containing any amount of a solute.,ecause distilled water is pure

and contains no dissolved substances+ an a1ueous solution with any concentration ofsolute will be hypertonic when compared to distilled water% "smosis is a process based

on the concentration of solute contained in two a1ueous solutions on either side of a

semipermeable membrane+ and is not dependent on the dissolved substance%

64! *!

K E*


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F':: &4S

@hen a1ueous solutions are separated by a semipermeable membrane+ the solution

containin the lesser concentration of solute+ the hypotonic solution+ will pass over to

the side containin the reater concentration of solute+ which is the hypertonic solution%

"nly the water will pass throuh the membrane+ leavin the solute behind%

"smotic pressure refers to the diffusion of water across a semipermeable membrane+

and is the result of two a1ueous solutions strivin toward a state of e1uilibrium% @hen

this state is reached+ the osmotic pressure is the same on both sides% This e1uali7ed

deree of osmotic pressure is called the hydrostatic+ or Nwater-stoppin+N pressure%

,ecause plant cells contain en7ymes+ salts and proteins dissolved in an a1ueous

solution+ a simple school lab eperiment can be used to demonstrate osmotic pressure%

lacin a stal. of celery in a bea.er of distilled water will cause the hypotonic water in

the bea.er to flow across the celery stal.'s cell membranes to e1uali7e the pressuredifference% The celery stal. will become riid as its cells fill with distilled water% lacin

another celery stal. in a bea.er containin a salt solution will cause the reverse effect%

The water leavin the hypotonic solution within the plant cells to cross over to the

hypertonic salt solution in the bea.er will cause the celery stal. to shrivel and become

limp%

http://www.ask.com/science/distilledwaterh)pertonich)potonic

+JF24992bedcJ99

http://www.br)nmawr.edu/biolog)/+ranklin/1.%onicit)02"02somlarit)$lab.pd+

2;* is considered denaturedwhen the double stranded 2;* moleculeis converted

into %%% GOC content of a 2;* moleculecan be estimated from itsthermal meltin %%%The more

dense the 2;* the 1uic.er it will sedimentupon centrifuation%

!ection 6%?urifyin+ 2etectin+ and Characteri7in roteins

&proteinmust be purified before its structure and the mechanism of its action can be studied.

However, because proteins vary in si4e, charge, and water solubility, no single method can be

used to isolate all proteins. /o isolate one particular protein from the estimated *5,555 different

proteins in a cell is a daunting task that re$uires methods both for separating proteins and for

detecting the presence of specific proteins.
http://www.ask.com/science/distilled-water-hypertonic-hypotonic-f87249092bedc899http://www.ask.com/science/distilled-water-hypertonic-hypotonic-f87249092bedc899http://www.brynmawr.edu/biology/franklin/1.Tonicity%20&%20Osomlarity_lab.pdfhttp://www.ncbi.nlm.nih.gov/books/n/mcb/A7315/def-item/A7752/http://www.ask.com/science/distilled-water-hypertonic-hypotonic-f87249092bedc899http://www.ask.com/science/distilled-water-hypertonic-hypotonic-f87249092bedc899http://www.brynmawr.edu/biology/franklin/1.Tonicity%20&%20Osomlarity_lab.pdfhttp://www.ncbi.nlm.nih.gov/books/n/mcb/A7315/def-item/A7752/


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&ny molecule, whetherprotein,carbohydrate, or nucleic acid, can be separated from other

molecules based on large differences in some physical characteristic. &lthough the se$uence of

amino acids in a protein uni$uely determines its function, the most useful physical characteristic

for separation of proteins issi%e,defined as either length or mass. In this section, we briefly

outline different techni$ues for separating proteins based on their si4e and other properties./hese techni$ues also apply to the separation of nucleic acids and other biomolecules. We then

consider general methods for detecting, or assaying,specific proteins, including the use of

radioactive compounds for tracking biological activity. 'inally, we discuss several techni$ues for

characteri4ing a protein2s mass, se$uence, and three#dimensional structure.

Go to:

roteins Can ,e


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&t very low concentrations, detergents dissolve in pure water as isolated molecules. &s the

concentration increases, the molecules begin to form micelles./hese are small, spherical

aggregates in which hydrophilicparts of the molecules face outward and the hydrophobicparts

cluster in the center (see'igure )#)5. /he critical micelle concentration (C&C)at which

micelles form is characteristic of each detergent and is a function of the structures of itshydrophobic and hydrophilic parts.

'onic detergentsbind to the exposed hydrophobicregions of membraneproteins as well as to the

hydrophobic core of water#soluble proteins. 9ecause of their charge, these detergents also disrupt

ionic and hydrogen bonds. &t high concentrations, for example, sodium dodecylsulfate

completely denatures proteins by binding to every side chain.onionic detergentsact in different

ways at different concentrations. &t high concentrations (above the +M+, they solubili4e

biological membranes by forming mixed micelles of detergent, phospholipid, and integral

membrane proteins ('igure :#:. &t low concentrations (below the +M+, these detergents may

bind to the hydrophobic regions of most membrane proteins, making them soluble in a$ueous

solution. In this case, although mixed micelles are not formed, the solubili4edproteinwill not

aggregate during subse$uent purification steps.

&iure 6-6>

"olubili4ation of integral membrane proteins by nonionic detergents. &t a concentration higher

than its critical micelle concentration (+M+, a detergent solubili4es lipids and integral

membrane proteins, forming mixed micelles containing(more...

Most peripheral proteins are bound to specific integral membraneproteins by ionic or other weak

i

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