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most widely used method for analyzing protein mixtures qualitatively. SDSPAGE separates proteins a ording to their mole ular weight! "ased on their differential rates of migration through a sieving matrix #a gel$ under the influen e of an applied ele tri al field and hen e an "e used to determine the relative mole ular mass of proteins. Samples to "e run on SDS- PAGE are firstly "oiled for % min in sample "uffer ontaining &mer aptoethanol and SDS. 'he mer aptoethanol #redu ing agent$ redu es any disulphide "ridges present that are holding together the protein tertiary stru ture and the SDS "inds strongly to and denatures the proteins. SDS #also alled lauryl sulfate$ is an anioni detergent that disrupts the se ondary! tertiary and quaternary stru ture of the protein to produ e a linear polypeptide hain oated with negatively harged SDS mole ules. (.)grams of SDS "inds per gram of protein or on average one SDS mole ule "inds every two amino a ids residues. 'herefore! the original native harges of the proteins are swamped "y negatively harged SDS mole ules and give every protein the same harge-to-mass ratio. *e ause the proteins have the same harge-to-mass ratio! and "e ause the gels have sieving properties! mo"ility "e omes a fun tion of mole ular weight. 'he sample "uffer also ontains a tra +ing dye "romophenol "lue that allows the ele trophoreti run to "e monitored! and su rose or gly erol! whi h gives the sample solution density thus allowing the sample to settle easily through the ele trophoresis "uffer to the "ottom when in,e ted into the loading well.
'he gels we will "e running use a dis ontinuous system! meaning that they have - parts. .ne is the separating gel! whi h has a high on entration of a rylamide and a ts as a mole ular sieve to separate the proteins a ording to size. *efore rea hing this gel! the proteins migrate through a sta +ing gel! whi h serves to ompress the proteins into a narrow "and so they all enter the separating gel at a"out the same time. 'he narrow starting "and in reases the resolution. 'his part of the gel has a lower on entration of a rylamide to avoid a sieving effe t. 'he sta +ing effe t is due to the gly ine in the "uffer! the low p/ in the sta +ing gel! and the higher p/ in the running "uffer. At the p/ of the sample and sta +ing gel #p/ 0.1$ gly ine is poorly ionized so that it2s effe tive mo"ility is very low! hloride ions have a mu h higher mo"ility at this p/ while mo"ilities of proteins are intermediate "etween the hloride and gly ine. 'he moment voltage is applied! the hloride ions #leading ions$ migrate away from the gly ine ions #the trailing ions$ leaving "ehind a zone of lower ondu tivity. Sin e ondu tivity is inversely proportional to field strength! this zone attains a higher voltage gradient whi h now a elerates gly ine mole ules so that it +eeps up with the hloride ions. A steady state is esta"lished where the produ ts of mo"ility and voltage gradient for gly ine and for hloride are equal and these harged spe ies now move at the same velo ity with a sharp "oundary "etween them. As this gly ine3 hloride "oundary
"is!< =!=>-methylene-"isa rylamide$.moves through the sample and the sta +ing gel! a low voltage gradient moves in front of the moving "oundary and a high voltage gradient "ehind it. @n the presen e of light and oxygen! ri"oflavin is onverted to its leu o form! whi h is a tive in initiating polymerization. 'herefore! the effe tive mo"ility of gly ine in reases so that the gly ine overta+es the proteins and now migrates dire tly "ehind the hloride ions. 'hese two effe ts ause the proteins to "e unsta +ed. 4hen the moving "oundary rea hes the interfa e of the sta +ing and resolving gels! the p/ of the gel in rease #p/ 1. 'his is usually referred to as photo hemi al polymerization. 'he proteins now move in the zone of uniform voltage gradient and p/ value and are separated a ording to their size. 'he proteins "ands an "e o"served "y staining the gel with 5oomassie *rilliant *lue 6-%7 followed "y washing in a destaining solution. Any proteins in front of the moving "oundary are rapidly overta+en sin e they have a lower velo ity than the hloride ions. At the same time! the gel pore size de reases there"y retarding the migration of the proteins "e ause of mole ular sieving. 'he relative mo"ility #6m$ of the protein "and an "e al ulated using the formula given "elow8 6m 9 Distan e moved "y protein "and::: Distan e moved "y the tra +ing dye echanism of Polymeri!ation Polya rylamide gels are formed "y opolymerization of a rylamide and "is-a rylamide #. *ehind the moving "oundary! in the higher voltage gradient! the proteins have a higher velo ity than gly ine.1$ mar+edly and this leads to a large in rease in the degree of disso iation of gly ine. 'he persulfate free radi als onvert a rylamide monomers to free radi als whi h rea t with una tivated monomers to "egin the polymerization hain rea tion. . Polymerization is initiated "y ammonium persulfate and 'E?ED #tetramethylethylenediamine$8 'E?ED a elerates the rate of formation of free radi als from persulfate and these in turn atalyze polymerization. 'he elongating polymer hains are randomly rosslin+ed "y "is! resulting in a gel with a hara teristi porosity whi h depends on the polymerization onditions and monomer on entrations. 'his property is parti ularly important! sin e ammonium persulfate "egins to "rea+ down almost immediately when dissolved in water. 'hus the moving "oundary sweeps up the proteins so that they "e ome on entrated into very thin zones or sta +s. 6i"oflavin #or ri"oflavin-%>-phosphate$ may also "e used as a sour e of free radi als! often in om"ination with 'E?ED and ammonium persulfate. 'his is why ammonium persulfate solutions should "e prepared fresh daily. 'he rea tion is a vinyl addition polymerization initiated "y a free radi al-generating system. Ammonium persulfate is also very hygros opi . 'herefore! the a umulation of water in ammonium persulfate results in a rapid loss of rea tivity.
/)0 'ris #hydroxymethyl$aminomethane 0.7 ml .-l.&<-'etramethyl ethylene diamine ('E ED) /) "eser6oir buffer ('ris-glycine.1 and the final volume was made to (77ml with water.0 g SDS SDS (.7 g 4as dissolved in water! the p/ ad.% ml Sample buffer +2 SDS ).usted to 0.&<.1 (-. p.( filter paper and stored at 7-)B5. p. 0)5) 10x Electrode (Running) Buffer.7 g 30. 5) "esol6ing gel buffer stoc* ('ris-. 0)0 'ris #hydroxymethyl$aminomethane A0.( filter paper and stored in "rown "ottle at 7-)B5. p.7 ml 'he p/ was ad.7 ml Gly erol -7. =) SDS ((78.) &. 1) (78 (9:6) Ammonium persulfate (APS) in 9ater8 Prepared "y dissolving (77mg of APS in (ml of water.ust "efore use.1!M Tris #$ %.1 12 "esol6ing gel buffer Gel 4uffer.8 0."E#$%"E E&'S: • • • Gel ele trophoreti unit from *io-6ad Power pa + ?i ropipette! Eppendorf tu"es! Sha+er! et "eagents () Acrylamide-bisacrylamide stoc* solution ( onomer stoc* solution) A rylamide A7. 'his reagent should "e prepared fresh .usted to 1. 0)0) (.usted to 1.( filter paper and stored at 7-)B5. . 'he solution was filtered through 4hatman =o.9:6): ( g of SDS #sodium lauryl sulfate$ was dissolved in (7ml of distilled water and stored at room temperature.7 ml 'he p/ was ad.% ? 'ris-/5l! p/ 0.7 g (? /5C )1.7g *isa rylamide 7. 0) Sample buffer +2 (? 'ris-/5l! p/ 0. /)0) 7.3 (makes 1 ) 'ris A. 'he solution was filtered through 4hatman =o.&. 'he solution was filtered through 4hatman =o. p.1 12 -3&-E&'"A'ED "unning Gel 4uffer. pH 8.A and final volume was made to ( C with water.% ? 'ris-/5l! p/ 1.3 g Tris base Gly ine ().) g 144.15 g/100 ml) (? /5C )1.-l.1 g 4as dissolved in water and final volume was made to (77ml.1 and the final volume was made to (77ml with water.04% *romophenol "lue an& 10% BME) &-?er aptoethanol BME (7. +) Stac*ing gel buffer stoc* ('ris-.A g (18. p.0 g Glycine 10.7 g (4% SDS !0% Glycer"l 0.
) / ) = ) A rylamide-"isa rylamide sto + solution#A7D'! -.-7 7. >) Staining solution 5oomassie "rilliant "lue 6--%7 (.A7 G.8) (ml of the solution) (-.%7 ------E.%? 'ris-/5l! p/ 1.(D *romophenol "lue ).7-7. =) 'he lid of the tan+ was losed and the gel was run at (77 volts until the "romophenol "lue dye rea hes near the "ottom of the gel sla".0E % -------7.G( 7.0ED 5$ )F Sta +ing gel "uffer sto + solution #7. +) 'he resolving gel prepared as per the ta"le! is then poured in "etween the plates avoiding any air "u""les entrapment.1$ (7D SDS (7D APS 4ater 'E?ED 'otal 6olume -.A7 7. 1) 'he sta +ing gel was prepared as per the ta"le! and was immediately poured on top of the resolving gel and the om"s were inserted into this gel.77 gel ((+). .A11 7.) After the sta +ing gel has polymerized! the om"s were removed and the gel plate was pla ed in the gel assettes assem"ly.-7 ((. 'he mixture was "oiled in a "oiling water "ath for A-% min and then ooled at room temperature.0 KDa$ was dissolved in (ml of the sample "uffer diluted with water in the ratio of (8(. (7) Destaining solution Gla ial a eti a id E% ml ?ethanol %7 ml 4ater was added to ma+e the final volume to ( C.%? 'ris-/5l! p/ 0. 5) 4hen the gel has polymerized! the water layer was arefully removed. Stac*ing gel and resol6ing gel preparation: *oth the gels for SDS-PAGE are prepared a ording to the parti ulars given in the ta"le "elow8 Stac*ing gel (18) Stoc* solution ( ) + ) 5 ) 1 ) . /) (7--7 Hl of the sample was loaded into ea h of the well.7(A7. 'he gel was allowed to polymerize.1$ )F 6esolving gel "uffer sto + solution #(. b) Preparation of gel and electrophoresis () 'he two glass plates are appropriately assem"led on the asting stand with the help of the asting frame. 'he gel assette assem"ly was pla ed into the ?ini tan+ and the reservoir "uffer was poured into inner ham"er and the tan+.7 ml 4ater was added to ma+e the final volume to (77ml. . BSA Mw=66.77 "esol6ing Procedure: a) Sample preparation (mg of the protein sample #Bovine serum albumin.-% g ?ethanol -77 ml Gla ial a eti a id A% ml 'he final volume was made to %77ml with distilled water and filtered to remove any undissolved material and stored at room temperature.% 7. Distilled water was then overlaid on top of the gel as gently as possi"le and the gel was allowed to set for A7-)%min.
. Destaining of the gel step is done the next day with the destaining solution till a lear "a +ground of the gel was o"tained. (7) 'he distan e travelled "y the dye and the various protein "ands were re orded and the 6m values al ulated.0) After the ele trophoresis is omplete! the power pa + is turned off and the gel is arefully removed from the gel plates. >) 'he gel is then pla ed in a tray ontaining the staining solution and stained overnight.
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