Aim: Separation of proteins by SDS PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) Principle: SDS-PAGE is the 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

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. Any proteins in front of the moving "oundary are rapidly overta+en sin e they have a lower velo ity than the hloride ions. *ehind the moving "oundary! in the higher voltage gradient! the proteins have a higher velo ity than gly ine. 'hus the moving "oundary sweeps up the proteins so that they "e ome on entrated into very thin zones or sta +s. 4hen the moving "oundary rea hes the interfa e of the sta +ing and resolving gels! the p/ of the gel in rease #p/ 1.1$ mar+edly and this leads to a large in rease in the degree of disso iation of gly ine. '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. At the same time! the gel pore size de reases there"y retarding the migration of the proteins "e ause of mole ular sieving. 'hese two effe ts ause the proteins to "e unsta +ed. '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. '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 #;"is!< =!=>-methylene-"isa rylamide$. 'he rea tion is a vinyl addition polymerization initiated "y a free radi al-generating system. Polymerization is initiated "y ammonium persulfate and 'E?ED #tetramethylethylenediamine$8 'E?ED a elerates the rate of formation of free radi als from persulfate and these in turn atalyze polymerization. 'he persulfate free radi als onvert a rylamide monomers to free radi als whi h rea t with una tivated monomers to "egin the polymerization hain rea tion. 'he elongating polymer hains are randomly rosslin+ed "y "is! resulting in a gel with a hara teristi porosity whi h depends on the polymerization onditions and monomer on entrations. 6i"oflavin #or ri"oflavin-%>-phosphate$ may also "e used as a sour e of free radi als! often in om"ination with 'E?ED and ammonium persulfate. @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. 'his is usually referred to as photo hemi al polymerization. Ammonium persulfate is also very hygros opi . 'his property is parti ularly important! sin e ammonium persulfate "egins to "rea+ down almost immediately when dissolved in water. 'herefore! the a umulation of water in ammonium persulfate results in a rapid loss of rea tivity. 'his is why ammonium persulfate solutions should "e prepared fresh daily.

"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 A7.7g *isa rylamide 7.1 g 4as dissolved in water and final volume was made to (77ml. 'he solution was filtered through 4hatman =o.( filter paper and stored in "rown "ottle at 7-)B5.
+) Stac*ing gel buffer stoc* ('ris-,-l. p, /)0) 7.% ? 'ris-/5l! p/ 0.1 12 -3&-E&'"A'ED "unning Gel 4uffer. p, /)0

'ris #hydroxymethyl$aminomethane 0.7 g (? /5C )1.7 ml 'he p/ was ad,usted to 0.1 and the final volume was made to (77ml with water. 'he solution was filtered through 4hatman =o.( filter paper and stored at 7-)B5.
5) "esol6ing gel buffer stoc* ('ris-,-l. p, 0)0) (.% ? 'ris-/5l! p/ 1.1 12 "esol6ing gel buffer Gel 4uffer. p, 0)0 'ris #hydroxymethyl$aminomethane A0.A g (18.15 g/100 ml) (? /5C )1.7 ml 'he p/ was ad,usted to 1.1 and the final volume was made to (77ml with water. 'he solution was filtered through 4hatman =o.( filter paper and stored at 7-)B5. 1) (78 (9:6) Ammonium persulfate (APS) in 9ater8 Prepared "y dissolving (77mg of APS in (ml of water. 'his reagent should "e prepared fresh ,ust "efore use. ;) &.&.&<.&<-'etramethyl ethylene diamine ('E ED) /) "eser6oir buffer ('ris-glycine. p, 0)5) 10x Electrode (Running) Buffer, pH 8.3 (makes 1 ) 'ris A.7 g 30.3 g Tris base Gly ine ().) g 144.0 g Glycine 10.0 g SDS SDS (.7 g 4as dissolved in water! the p/ ad,usted to 1.A and final volume was made to ( C with water. =) SDS ((78.9:6): ( g of SDS #sodium lauryl sulfate$ was dissolved in (7ml of distilled water and stored at room temperature. 0) Sample buffer +2 (? 'ris-/5l! p/ 0.1 (-.% ml Sample buffer +2 SDS ).7 g (4% SDS !0% Glycer"l 0.1!M Tris #$ %.8 0.04% *romophenol "lue an& 10% BME) &-?er aptoethanol BME (7.7 ml Gly erol -7.7 ml

(D *romophenol "lue ).7 ml 4ater was added to ma+e the final volume to (77ml. >) Staining solution 5oomassie "rilliant "lue 6--%7 (.-% 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) Destaining solution Gla ial a eti a id E% ml ?ethanol %7 ml 4ater was added to ma+e the final volume to ( C.

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 ) ; ) / ) = ) A rylamide-"isa rylamide sto + solution#A7D'! -.0ED 5$ )F Sta +ing gel "uffer sto + solution #7.%? 'ris-/5l! p/ 0.1$ )F 6esolving gel "uffer sto + solution #(.%? 'ris-/5l! p/ 1.1$ (7D SDS (7D APS 4ater 'E?ED 'otal 6olume -.0E % -------7.-7 7.-7 ((.G( 7.7-7.77 gel ((+);8) (ml of the solution) (-.%7 ------E.% 7.A7 7.A7 G.A11 7.7(A7.77 "esol6ing

Procedure: a) Sample preparation (mg of the protein sample #Bovine serum albumin, BSA Mw=66.0 KDa$ was dissolved in (ml of the sample "uffer diluted with water in the ratio of (8(. 'he mixture was "oiled in a "oiling water "ath for A-% min and then ooled at room temperature. 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 resolving gel prepared as per the ta"le! is then poured in "etween the plates avoiding any air "u""les entrapment. 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. 5) 4hen the gel has polymerized! the water layer was arefully removed. 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. 'he gel was allowed to polymerize. ;) 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. '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--7 Hl of the sample was loaded into ea h of the well. =) '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".

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. 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.