This molecular sieving separates molecules on the basis of size. Molecules can also be separated on the basis of their net charge. Separation on the basis of charge requires the use of an unrestrictive matrix of large pore size, such as agarose. Most of the early research in protein electrophoresis was done with gels made of potato starch. Since the 1960s, polyacrylamide has all but replaced starch as the medium for protein separations.
Researchers can vary the size of the pores by adjusting the concentration of acrylamide.
Since proteins come in all sizes and shapes, varying the pore size allows researchers to tailor their gel to best fit their experimental needs. Samples are treated with SDS, an anionic detergent, in the presence of a reducing agent such as b-mercaptoethanol. This treatment unfolds the protein into a linear molecule, uniformly coated with negative charges. When electrophoresed, the proteins travel through the gel toward the anode and are separated solely on the basis of their molecular size. The separated proteins are then compared with size standards to estimate molecular weight.