Electrophoresis that is carried out in polyacrylamide gel is called polyacrylamide gel electrophoresis. Crosslinked polyacrylamide gel is formed by polymerization of monomer acrylamide in the presence of bisacrylamide. Bisacrylamide consists of two molecules of acrylamide linked by methylene group. Thus it is used as crosslinking agent. Polymerization of monomer acrylamide takes place in head to tail fashion and long chain is formed. This polymerization is an example of free radical catalysis. Polymerization is initiated by addition of ammoniumpersulfate and N, N, N’, N’-tetramethyl ethylene diamine (TEMED) and sodium dodecyl sulfate (SDS) or sodium lauryl sulfate. TEMED catalyses the decomposition of persulfate ion into free radical. This free radical is highly reactive due to the presence of unpaired electron and need to be paired with another electron to stabilize the molecule. Thus long chained polyacrylamide gel is formed. Polymerization is exothermic. Heat generated during polymerization warms gel and removes oxygen bubbles trapped in gel. Oxygen interferes with polymerization. Therefore, degassing is done before polymerization.
Polymerization is generally used in concentration between 3-30%. Pore size is determined by concentration of both acrylamide and bisacrylamide.
Acrylamide and bisacrylamide are neurotoxins. Although, polyacrylamide is not toxic, it may still contain some molecules of acrylamide and bisacrylamide that are not polymerized. Therefore, care should be taken and gloves be used to handle.
SDS is an anionic detergent which binds with most proteins in amount proportional to molecular weight of protein. One SDS molecule binds with two amino acid residues. Bound SDS contributes large amount of negative charge rendering intrinsic charge of protein insignificant and all proteins have similar charge to mass ratio. In addition, native conformation of protein is altered and opened into rod shaped structure and all proteins have similar shape. Therefore, electrophoresis in the presence of SDS is exclusively based on molecular weight of protein.
Sample to be run on SDS-PAGE is boiled for 5 minutes in sample buffer containing β-mercaptoethanol and SDS. β-mercaptoethanol reduces disulphide bridge present holding protein tertiary structure and SDS denatures and binds protein. Any protein in sample is denatured by this step with a series of negatively charged SDS along the polypeptide chain. Sample buffer also contains tracking dye bromophenol blue to monitor electrophoresis run and sucrose or glycerol that increases density allowing sample to settle down at bottom through electrophoresis buffer when injected into loading well. Once all samples are loaded, current is passed through gel. Actually, sample is not directly loaded on main separating gel but is loaded on stacking gel. Main separating gel solution is poured between glass plates and after solidification, thin layer of stacking gel is poured in which loading well is formed. Stacking gel is to concentrate protein into sharp band before entering separating gel. It is based on phenomena: isotachophoresis. Pore size in stacking gel is large and protein is stacked. Bromophenol blue (BROMOPHENOL BLUE INDICATOR 100ML
) is small molecule. So it moves faster than sample protein and is used as tracking dye to monitor electrophoretic font. Once this front reaches bottom of gel, current is removed. Gel is removed and dipped in staining solution of Coomassie Brilliant Blue. Acid: Methanol in staining solution cause denatured protein to precipitate or fix and prevents from being removed from gel during washing. Destaining solution removes background dye from gel.In SDS PAGE (Analysis of protease digestion patterns in tideland sediments using SDS-PAGE [An article from: Journal of Experimental Marine Biology and Ecology]
), a single pure protein gives single band unless it is composed of two unequal subunits.Molecular weight of protein can be determined by comparing its mobility with that of other standard protein with known molecular weight.
