蛋白酶抑制剂的配制
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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:
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.
Activation of Sodium Orthovanadate (sigma s-6508)
Sodium orthovanadate should be activated for maximal inhibition of protein phosphotyrosyl-phosphatases .
1. Prepare powder needed for 200 mM solution of sodium orthovanadate,
dissolve in ddH2O.
2. 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.
3. Boil the solution until it turns colorless (approximately 10 minutes).
4. Cool to room temperature.
5. Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution
remains colorless and the pH stabilizes at 10.0.
6. adjust volume.
7. 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 phosphatases. Please note that adding DTT rapidly inactivates sodium orthovanadate.
Sodium fluoride (Sigma, s-1504)
Dissolve sodium fluoride to 500mM in ddH20, store in darkness at 4 degree. Also you first make activated 400 mM sodium orthovanadata and 1 M sodium fluoride separately, and then 1:1 mix them together to a 100x concentration (200mM sodium orthovanadata and 500 mM sodium fluoride). Aliquot to 15ml tubes, keep in -20°C and in darkness.