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