Sample preparation for Western Blot

There are different methods of sample preparation depending on whether a protein is extracellular or intracellular. Intracellular proteins are more difficult to extract, and its procedure must be carefully devised.

Figure 1. Sample preparation for Western Blot.

Every organism’s cellular make-up is comprised of a complex mixture of molecules including proteins, nucleic acids, polysaccharides, and salt. These molecules can interfere with the Western blot method, hence the importance of sample preparation. There are numerous cell disruption methods to prepare the sample for Western blotting. Typically when dealing with mammalian cells, you can remove the cell membrane by detergent lysis, freeze/thaw lysis or mechanical homogenization and sonication.

After the extraction, the protein sample is boiled in a solution containing tracking dye (bromophenol blue), disulfide reducing agent (beta-mercaptoethanol), and detergent (sodium dodecyl sulfate/ SDS). Boiling denatures the protein, thereby unfolding it. Beta-mercaptoetanol prevents reformation of disulfide bonds and thus disrupting the quaternary and tertiary structure of the protein, creating linear chain polypeptides. Sodium dodecyl sulfate confines the protein with a negative charge.