I am purifying a disorder protein (8 kDa) but during binding to Ni-NTA column it got degraded. Currently I am using Benzamidine (2 mM) as a protease inhibitor. can I use PMSF and Benzamidine together in lysis buffer? If yes Please suggest the conc. of both.
As @Craig says, it is good to know what your system holds in store. Initial tests can be very helpful and save time and money. As @Arshad mentions, it is also good to know something about the mechanism of the inhibition. Benzamidine is a reversible (!) competitive inhibitor of serine proteinases. If effective you will need to include it at every step until the proteinases are gone. PMSF also inhibits serine proteases (irreversibly!) and the cysteine protease papain (reversible by DTT treatment). Using both is a bit of belt and suspenders, but if found to be more effective than PMSF alone, you have your answer.
If you are working with a cell extract, you probably can't be 100% sure about what protease is causing the damage, or perhaps if there are multiple enzymes involved. You might look into a broad spectrum protease inhibitor cocktail. There are variations sold by some of the big chemical vendors, specifically meant for use with cell and tissue extracts. It might be easier to aggressively inhibit, if your only goal is to purify without further complications.
As @Craig says, it is good to know what your system holds in store. Initial tests can be very helpful and save time and money. As @Arshad mentions, it is also good to know something about the mechanism of the inhibition. Benzamidine is a reversible (!) competitive inhibitor of serine proteinases. If effective you will need to include it at every step until the proteinases are gone. PMSF also inhibits serine proteases (irreversibly!) and the cysteine protease papain (reversible by DTT treatment). Using both is a bit of belt and suspenders, but if found to be more effective than PMSF alone, you have your answer.
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