蛋白酶抑制剂的配制.docVIP

How to make protease and phosphotase inhibitors Protease inhibitors are rapidly inactivated in aqueous solutions and should be added to solutions immediately before use. Cocktail solutions are stored at -20 degrees and are good for approximately 6 months. I. Protease Inhibitor Cocktail The Protease inhibitor cocktail is used as 100X for extract preps and as 1000X for column elution buffers. Cocktail is made in 100% EtOH and contains: inhbitor Sigma catalog # powder storage concentration in cocktail amt for 25 mls cocktail Aprotinin* A1153 4 deg C 1 mg/ml 25 mg Pepstatin A* P5318 4 deg C 1 mg/ml 25 mg Leupeptin* L2884 -20 deg C 1 mg/ml 25 mg Some components are not completely soluble in 100% EtOH. Vortex solution well before use to obtain a cloudy suspension and add to buffer - they become soluble in aqueous II. 100 mM PMSF PMSF is very unstable in aqueous solutions. The 100 mM stock solution is made in isopropanol and stored in the dark at -20 degrees. Must be warmed 37 degrees to get into solution. Use at 1 mM in solutions. inhbitor Sigma catalog # powder storage concentration amt for 25 mls PMSF P7626 room temp 100 mM 440 mg Activation of Sodium Orthovanadate sigma s-6508 Sodium orthovanadate should be activated for maximal inhibition of protein phosphotyrosyl-phosphatases . Prepare powder needed for 200 mM solution of sodium orthovanadate, dissolve in ddH2O. Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow. Boil the solution until it turns colorless approximately 10 minutes . Cool to room temperature. Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0. adjust volume. Store the activated sodium orthovanadate as aliquots at -20°C. This procedure depolymerizes the vanadate, converting it into a more potent inhibitor of protein tyrosine phospha