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 |
compiled by Chad Rappleye
Aroian Lab Protocols