As proteins are macromolecules which bear an overall charge at a particular pH, they may be separated on the basis of size through gel electrophoresis. However the different chemical nature of proteins to that of nucleic acids means that the techniques and equipment used are slightly different.

Proteins have a tertiary structure which is held together through hydrogen bonding between amino acid side chains and sometimes covalent bonding through disulphide bridges between cysteine residues. This tertiary structure will affect the migration of the protein through the gel. (ie. a tightly wound globular protein will migrate differently to a long fibrous protein, even though the two may be of similar size). Therefore proteins must be denatured through heating and treatment with a detergent (SDS or sodium dodecyl sulphate) and treatment with a reducing agent which breaks the disulphide bridges (eg. β-Mercaptoethanol).

Protein gels are composed of polyacrylamide (hence polyacrylamide gel electrophoresis, or PAGE). Unlike agarose gels which are heated to dissolve the agarose and then set upon cooling, the polymerization of acrylamide into polyacrylamide is a chemical process triggered by the compound N, N, N’, N’-tetraethylenediamine (TEMED). The gels are cast between two glass plates separated by spacers and as such are generally much thinner than agarose gels for DNA.

PAGE gels are also cast in two sections. The top section, where the wells are made and samples loaded is called the stacking gel and is generally around 3.5-4% polyacrylamide and pH 6.8. In this gel, the proteins form discrete bands which improve the resolution of the overall gel. The lower section is the resolving gel and ranges from 4-20% polyacrylamide (depending on the proteins investigated) and pH 8.8. It is in the resolving gel that the protein bands are analysed.


As with DNA gel electrophoresisprotein samples are run against standards containing proteins of known sizes. While the band migration in DNA gel electrophoresis is expressed by comparison to fragments of DNA containing different lengths of nucleotide bases (bases or kilobases), protein standards are expressed in terms of kilodaltons (kDa), where one kilodalton is equivalent to the mass of 1000 hydrogen atoms.

Proteins are normally visualized using staining (dyes such as Coomassie Blue, or silver staining methods). However more specific identification of proteins may be made using immunoblotting techniques. In these techniques, the protein bands are transferred to a strip of cellulose acetate and incubated with antibodies to the protein labeled with a compound which produces a colour when treated chemically. The antibodies and their labels bind only to the bands on the cellulose acetate containing the protein of interest, resulting in coloured bands in those regions.

The following videos may be useful in understanding the techniques involved in SDS-PAGE (Note : These are all You Tube videos and are hosted on an external site. Some users may not be able to access them from certain computers) :