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

SDS solubilizes the proteins to give them uniform negative charge, hence the separation is based on size or molecular weight. It helps in stabilizing the gel matrix for good resolution during the electrophoresis. It also increases the pH of the gel to separate multiunit proteins into single unit.

SDS is a detergent which along with a bit of boiling and a reducing agent disrupts the tertiary structure of protein (brings the folded proteins down to linear molecules). Boiling leads to disruption of tertiary structure of proteins (So protein solution is heat denatured prior to gel loading). SDS binds uniformly to the linear proteins, which means that the charge of the protein is now approximately proportional to its molecular weight.