ABSTRACT

Throughout the purification process the purity of the pooled target protein fraction should be monitored regularly; denaturing polyacrylamide gel electrophoresis (PAGE) is the most widely used technique to monitor the purity of pooled protein fractions after chromatography. This technique denatures proteins with a detergent (sodium dodecyl sulfate (SDS)) and a reducing agent (e.g. 2-mercaptoethanol (2-ME); Figure 6.1) into polypeptides and then separates the polypeptides according to their relative molecular mass (Mr). A single band on an SDS-PAGE gel is a good indication of a homogeneous preparation of protein. However, because SDS-PAGE separates polypeptides on their Mr, a single band does not rule out the possibility that there are other polypeptides present in the sample with the same Mr. To verify the homogeneity of the protein preparation, alternative methods can be used in combination with denaturing SDS-PAGE. These supplementary techniques must exploit a physical parameter other than the molecular mass of proteins to increase the stringency of the analysis. For example, PAGE in the absence of a detergent (nondenaturing PAGE) will separate proteins according to both their hydrodynamic volume (related to Mr) and surface charge. A homogeneous band on both denaturing and nondenaturing PAGE is a good indication of the purity of the protein fraction. Other techniques which could be considered to supplement denaturing SDS-PAGE include: isoelectric focusing (IEF), capillary electrophoresis (CE), or reversed phase chromatography (RPC).