Protease Inhibitors

from Alberts" Lab Protocols (Kathy Miller/Chris Field)

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
Benzamidine HCL	B6506	4 deg C	1.6 mg/ml	40 mg
Phenanthroline	P1294	room temp	1 mg/ml	25 mg
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

* inhibitors purchased in one-time use amounts

** 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