**2.3.8 MW estimation**

The migration rate of a polypeptide in SDS-PAGE is inversely proportional to the logarithm of its MW. The larger the polypeptide, the more slowly it migrates in a gel. MW is determined in SDS-PAGE by comparing the migration of protein spots to the migration of standards. Plots of log MW versus the migration distance are reasonably linear. Gradient SDS-PAGE gels can also be used to estimate MW. In this case, log MW is proportional to log (%T). With linear gradients, %T is proportional to distance migrated, so the data can be plotted as log MW vs. log (migration distance). Standard curves are actually sigmoid. The apparent linearit of a standard curve may not cover the full MW range for a given protein mixture in a particular gel. However, log MW varies sufficiently slowly to allow accurate MW estimates to be made by interpolation, and even extrapolation, over relatively wide ranges (Garfin 1995). Mixtures of standard proteins with known MW are available from Bio-Rad in several formats for calibrating the migration of proteins in electrophoretic gels. Standards are available unstained, prestained, or with tags for development with various secondary reagents (useful when blotting). Standards can be run in a reference well, attached to the end of a focused IPG strip by filter paper, or directly embedded in agarose onto the second-dimension gel

#### **2.4 Detection of proteins in gels**
