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:

• Sodium dodecyl sulfate(SDS) for breaking bonds and creating a uniformly negative charge
• Beta mercaptoethanol for cleaving disulfide bonds in particular
• Bromophenol blue as an indicator for migration through the gel
• Glycerol to weigh the sample down
• Hydrochloric acid (HCL) to ionize the sample for electrophoretic mobility

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.

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