32.12: SDS-PAGE
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges and size, as seen in native PAGE. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only, as seen in SDS-PAGE. Polypeptide chains can migrate in polyacrylamide gel even when coated with SDS. So, with this observation, Ulrich K. Laemmli developed the technique of SDS-PAGE in the year 1970.
In SDS-PAGE, the polyacrylamide gels are formed by the polymerization of acrylamide monomers that are transparent, chemically and biologically inert, and uncharged. These gels have a controllable pore size determined during gel preparation, where the concentration of acrylamide and bisacrylamide, the cross-linking agent, regulate the gel’s pore size. Acrylamide monomers thus polymerize to form polyacrylamide, wherein ammonium persulfate catalyzes the polymerization reaction. SDS provides a uniform charge to mass ratio for all proteins. Usually, a gram of protein is covered by 1.4 gm of SDS allowing size-driven protein separation.
The sample preparation buffer for SDS-PAGE, besides SDS and β-mercaptoethanol, contains glycerol and bromophenol blue. The density of glycerol helps the sample reach the bottom of the stacking well, preventing it from flowing out from the well into the buffer. Bromophenol blue functions as the tracking dye and indicates the proteins’ progress in the gel.
Optimum pH for each buffer solution is crucial as it determines ion concentration in the buffer required for protein movement under voltage application. The ionic strength and pH of the buffer used for running the gel (pH 8.3) is different from the buffers used to create the stacking gel (pH 6.8) and the resolving gel (pH 8.3). This pH difference between the stacking and resolving gel ensures that the low ionic strength of the stacking gel offers high electrical resistance, allowing the proteins to stack and then separate once they enter the resolving gel.
SDS-PAGE has varied applications, including the estimation of protein size, purity, and even peptide mapping. The major limitation of this technique is that it cannot obtain information about the enzyme activity, cofactor, and protein binding interactions. It is challenging to analyze highly acidic or basic proteins using SDS-PAGE.
