Molecular weight determination By using SDS-PAGE

Introduction Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a reliable method for determining the molecular weight (MW) of an unknown protein. The first step in MW determination of a protein is to separate the protein sample on the same gel with a set of MW standards. Next, a graph of log MW vs. relative migration distance (Rf) is plotted, based on the values obtained for the bands in the MW standard. The MW of the unknown protein band is then calculated by interpolation using this graph. The key to determining MW accurately is selecting separation conditions that will produce a linear relationship between log MW and migration within the likely MW range of the unknown protein.

Some Factors to Consider The standard protein and the unknown protein should be electrophoresed on the same gel under identical conditions. Multiple data points should be generated to make sure that the data carries a statistical significance. For best results, try using at least three gels. In solubilizing the proteins, the sample buffer should contain reducing agents such as dithiothreitol or B- mercaptoethanol to ensure that the disulfide bonds will be broken. As you may already know, disulfide bonds reduce the effect of secondary structure on migration. The sample buffer should also contain SDS. SDS binds to hydrophobic protein regions to denature secondary, tertiary and quaternary structures and give a net negative charge on the proteins. SDS causes proteins to unfold to random, rod-like chains without breaking any covalent bonds in the process. This causes proteins to lose their biological functions without damaging their primary structures.

Some Factors to Consider For accurate MW determination, the unknown protein should be within the linear range of the standard curve, and the amount of the unknown protein (or its intensity after staining) should match the corresponding standard .

MW vs RF

LogMW VS Rf

LogMW vs Rf The extreme points were removed

Rf determination

Rf determination

Continuous vs discontinuous

gradient gel vs Single-percentage gels (gradient gel)  if you want to separate a complex of proteins of different M.Wts need different percentages of acrylamide i.e Gradient PAGE (4-20%) resolve a much wider size range of proteins on a single gel

gradient gel vs Single-percentage gels (Single-percentage gels ) If you know the protein of interest (M.Wt), you can select a particular percentage of stacking PAGE Easy to perform

gradient gel vs Single-percentage gels

Prestained vs unstained std protein Prestained std protein Monitoring the separation process Monitor the transfer process on WB Provide materials used in WB

Prestained vs unstained std protein Unstained std protein Proffered in molecular weight determination (prestained markers do not provide the same size precision as unstained proteins. The apparent molecular weight of a prestained protein is usually greater than the unstained protein) Detection error in stain or sample preparation

Limitations The presence of polypeptides such as glycol- and lipoproteins usually leads to erroneous results since they are not fully coated with SDS and thus, would not behave as expected.  factors such as protein structure, posttranslational modifications, and amino acid composition are variables that are difficult or impossible to minimize and can affect the electrophoretic migration.

Limitations Glycoproteins migrate unpredictably in SDS-PAGE (Hames 1998). The hydrophilic glycan moieties can obstruct the binding of SDS, and the decreased hydrophobic interaction between the protein and SDS result in an inconsistent chargeto-mass ratio. However, some evidence suggests that glycoproteins exhibit more normal protein migration in gradient gels.

Limitations Acidic proteins (such as tropomyosin) also migrate abnormally on SDS-PAGE gels. The acidic residues may be repelled by the negatively charged SDS, leading to an unusual mass-to-charge ratio and migration. Highly basic proteins (for example, lysozyme, histones, and troponin I), which contain an abundance of positively charged amino acids, migrate more slowly in SDS-PAGE due to a reduced charge-to-mass ratio, resulting in a higher apparent MW

Limitations Proteins with high proline content or with other unusual amino acid sequences (for example, ventricular myosin light chain) show a decreased electrophoretic mobility as a result of kinks and structural rigidity caused by the primary sequence.

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