Question

What chemical properties of sodium dodecyl sulfate (SDS) contribute to equal charge/mass ratios needed for molecular mass determinations using SDS-PAGE? Why is the protein solution heat denatured prior to gel loading?

Answer 1

Ans. SDS is a negatively charge amphipathic detergent. Its hydrophobic tail uniformly binds to the hydrophobic regions of a peptide chain whereas the negatively charged polar head simultaneously binds to the polar regions of peptide chain. Once added to a protein solution, it binds to all polypeptides chains and provide them uniform negative charge i.e. all polypeptide chains bear almost equal negative charge per unit length. Uniform negative charge also linearizes (primary structure of protein, no secondary of tertiary structures) the polypeptide. Therefore, all types of polypeptide have similar charge density and shape but differ only in their amino acid sequence i.e. also differ in their molecular masses. The polypeptides with different molecular mass move through the gel with different rate, the smallest being fastest and the largest being the slowest, and get resolved during electrophoresis. The comparison of Rf values of various proteins with the standard molecular mass markers provides an accurate method to determine the molar mass of the proteins.

# Heat treatment disrupts non-covalent interactions like H-bonds, van der Waal’s interactions, ionic interactions, etc. that further leads to protein denaturation. Denaturation facilitates better binding of SDS molecules along the peptide chain in relatively short time. Moreover, heat treatment also diminishes the interactions between protein and non-specific, non-protein molecules in the sample, thus frees protein from debris in solution. It also reduces the viscosity of the solution and makes sample loading in the wells more easier.