electrophoresis, the gel is put into a renaturing buffer in order to return the cathepsins to their native conformation. The gel is then put into an activation buffer of a specific pH and left to incubate overnight at 37 °C. This activation step allows the cathepsins to degrade the gelatin substrate. When the gel is stained using a Coomassie blue stain, areas of the gel still containing gelatin appear blue. The areas of the gel where cathepsins were active appear as white bands. This cathepsin zymography protocol has been used to detect femtomole quantities of mature cathepsin K. The different cathepsins can be identified based on their migration distance due to their molecular weights: cathepsin K (~37 kDa), V (~35 kDa), S (~25kDa), and L (~20 kDa). Cathepsins have specific pH levels at which they have optimum proteolytic activity. Cathepsin K is able to degrade gelatin at pH 7 and 8, but these pH levels do not allow for cathepsins L and V activity. At a pH 4 cathepsin V is active, but cathepsin K is not. Adjusting the pH of the activation buffer can allow for further identification of cathepsin types. History The term cathepsin was coined in 1929 by Richard Willstätter and Eugen Bamann to describe a proteolytic activity of leukocytes and tissues at slightly acidic pH (Willstätter & Bamann (1929) Hoppe-Seylers Z. Physiol. Chemie 180, 127-143). The earliest record of "cathepsin" found in the MEDLINE database (e.g., via PubMed) is from the Journal of Biological Chemistry in 1949. However, references within this article indicate that cathepsins were first identified and named around the turn of the 20th century. Much of this earlier work was done in the laboratory of Max Bergmann, who spent the first several decades of the century defining these proteases. It is notable that research published in the 1930s (primarily by Bergmann) used the term "catheptic enzymes" to refer to a broad family of proteases that included papain, bromelin, and cathepsin itself. Initial efforts to purify and characterize proteases using hemoglobin transpired at a time when the word "cathepsin" indicated a single enzyme; the existence of multiple, distinct cathepsin family members (e.g. B, H, L) did not appear to be understood at the time. However, by 1937 Bergmann and colleagues began to differentiate cathepsins on the basis of their source in the human body (e.g. liver cathepsin, spleen cathepsin). References External links The MEROPS