Question

what information give SDS-PAGE about the proteins that are being analysed ?

Answer 1

SDS-PAGE is a reliable method for determining the molecular weight (MW) of an unknown protein, since the migration rate of a protein coated with SDS is inversely proportional to the logarithm of its MW. The key to accurate MW determination is selecting separation conditions that produce a linear relationship between log MW and migration within the likely MW range of the unknown protein

To ensure accurate MW determination:

• Separate the protein sample on the same gel with a set of MW standards (see Protein Standards for information regarding selection of protein standards)
• For statistical significance, generate multiple data points (>3 gels)
• Use a sample buffer containing reducing agents (DTT or β-ME) to break disulfide bonds and minimize the effect of tertiary structure on migration
• Include SDS in the sample buffer. SDS denatures secondary, tertiary, and quaternary structures by binding to hydrophobic protein regions, and it confers a net negative charge on the proteins, which results in a constant charge-to-mass ratio; SDS binds proteins in the proportion of 1.4 g SDS/g protein

After separation, determine the relative migration distance (R_f) of the protein standards and of the unknown protein. Rf is defined as the mobility of a protein divided by the mobility of the ion front. Because the ion front can be difficult to locate, mobilities are normalized to the tracking dye that migrates only slightly behind the ion front:

R_f = (distance to band)/(distance to dye front)

Using the values obtained for the protein standards, plot a graph of log MW vs. R_f (see figure below). The plot should be linear for most proteins, provided they are fully denatured and that the gel percentage is appropriate for the MW range of the sample. The standard curve is sigmoid at extreme MW values because at high MW, the sieving effect of the matrix is so large that molecules are unable to penetrate the gel; at low MW, the sieving effect is negligible and proteins migrate almost freely.