Preparing protein samples for electrophoresis

A protein consists of one or more polypeptides and/or additional molecules that are held together by molecular interactions, including hydrogen bonds, disulphide bonds, hydrophobic interactions and ionic bonds. These molecular interactions create an organization within the structure of a protein. The primary level of organization is where each amino acid is connected to the next by a single peptide bond, creating a single-stranded chain called a polypeptide. Polypeptides then have additional interactions that result in levels of organizations that can be primary, secondary, tertiary, or quaternary. The organization is important for electrophoresis because the proteins must be in a primary structure to be separated by their molecular weight. The SDS procedure for sample preparation ensures that proteins reach their primary structure.

To unfold the protein into its primary structure, a loading buffer is added to the protein sample prior to running electrophoresis. The loading buffer contains:

After adding the buffer, the sample is heated. This ensures that the SDS binds fully to the protein, coating it in a negative charge. It also ensures that all bonds have been cleaved leaving the protein in its simplest primary structure.

Illustration of a protein before and after the influence of sodium dodecyl sulfate and beta-mercaptoethanol. To the left is an unaffected protein in its original structure that is held together in a folded formation by hydrogen, ionic and disulphide bonds together with hydrophobic interactions. Next is the influenced protein in a slightly unfolded structure where all hydrogen bonds, ionic bonds, disulphide bonds and hydrophobic interactions have been cleaved by SDS and beta-mercaptoethanol. Also the protein has been covered in negative charge.

Figure 1. Preparing proteins for SDS-PAGE: Effect of SDS and beta-mercaptoethanol on proteins.