Electrophoresis is a very common technology used in many different types of protein analysis, no matter if the protein of interest is purified or part of a complex sample. When optimizing conditions for expression of recombinant proteins, electrophoresis can be used to obtain information about protein yield at various conditions. Additionally electrophoresis can be used subsequently to purification by gel filtration, to verify protein purity and to confirm that the purified protein has correct molecular weight.
Electrophoresis of proteins is usually carried out by loading a sample into a well, to which a voltage is then applied; the varying size, shape and charge of molecules makes them move through the matrix at different velocities. At the end of the separation, the proteins are detectable as bands at different positions in the matrix.
Gel electrophoresis, as a means to separate proteins, is usually performed under denaturing conditions imparted by the presence of the detergent, SDS, both in the sample and as a constituent of the gel and running buffer. 1.4 g of SDS will bind to each gram of protein, so that any inherent charge on the protein is masked by the coating of negatively charged detergent micelles. Denaturing gels can be run under reducing conditions, where a reducing agent such as dithiothreitol (DTT) or β-mercaptoethanol is added to the sample buffer and heated. These reagents act by cleaving disulfide bonds between cysteine residues to disrupt the quaternary and tertiary structure of the proteins, creating linear chains of polypeptides. Proteins treated in this way migrate at rates that are a linear function of the logarithm of their molecular weights.
Alternatively, denaturing gels can be run under non-reducing conditions (no sample boiling and no added reducing agent) when it is important to maintain the native structure of proteins for further analysis.
Polyacrylamide gels, both as homogenous and as gradient, are the most commonly used matrices in for separation of proteins. In a complex sample where separation is desired over a wide range of molecular weights, a gradient gel with increasing gel density should be used. In such a gel, over a given time, small proteins will reach dense regions of the gel while larger proteins will migrate within less dense regions.
Hopefully that brief of electrophoresis was helpful but for more detail and information on its use please download our free handbook guide to protein purification or ask a question via the comments section below. Thanks for reading
