The Protein Man's Blog | A Discussion of Protein Research

While proteolytic enzymes such as proteases and phosphatases play an important role in living cells and help ensure the survival of the organism, the mechanisms that regulate the tightly controlled cellular environment is disrupted during cell lysis. When this happens, these enzymes may start cleaving a variety of proteins that they would otherwise not touch in intact cells. This situation often leads to reduced recovery of total protein and biologically meaningless representation of protein activities.

Proteases are enzymes that facilitate proteolysis, or the breakdown of protein and peptide molecules into smaller polypeptides and/or amino acids. These enzymes do their job by cleaving the peptide bonds linking the amino acids together in the polypeptide chains.

Dialysis is a solution-based separation technique used to facilitate the removal of small, unwanted compounds from macromolecules such as proteins, DNA, or polysaccharides through a semi-permeable membrane using the principle of selective diffusion. Although there are a lot of factors that affect the dialysis rate, the semi-permeable membrane is the single most important factor that can determine the success of the process.

After the protein bands have been separated by electrophoresis, researchers perform a dye-binding or color-producing chemical reaction to visualize the proteins embedded within the pores of the polyacrylamide gel matrix. Some of the most popular in-gel detection methods include staining with Coomassie dye, metallic silver, and zinc.