Tissues and cells often contain large amounts of proteases. During lysis, these are released and, in turn, can digest the target protein. Therefore, protease inhibitors are critical for preserving the target protein. Common protease inhibitors are PMSF (phenylmethylsulfonyl fluoride), Aprotinin, Leupeptin, Pepstatin, and AEBSF-HCL (4-benzenesulfonyl fluoride hydrochloride). PMSF is highly effective and is the most popular choice for lysate preparation. Many protease inhibitors require a divalent metal ion to function, so a sequestering agent is also often used to inhibit protease activity, such as EDTA. In addition, for phosphorylated target proteins, a phosphatase inhibitor such as sodium fluoride or sodium orthovanadate is needed to preserve the phosphorylated form of the protein. Sodium orthovanadate, in particular, is very effective, but needs to be activated by adjusting the pH of the solution to 10 and then boiling it until the solution is colorless. Other phosphatase inhibitors include sodium pyrophosphate and & beta-glycerol phosphate.
