Phenylmethylsulfonyl fluoride (PMSF) is an inactivator of serine proteases. It chemically modifies the thiol group in the active centre, so the proteases are send to kingdom come.

Benzamidine is a competitive inhibitor of serine proteases. In theory, it should not be required after PMSF, but only in theory  is there no difference between theory and practice.

So, in short, removing these substances from your homogenization buffer will not per se degrade your protein, but will give proteases (which are present everywhere) the chance to do so. 

Your yield may increase further if you add inhibitors or inactivators of metallo-proteases (EGTA, EDTA), Asp-proteases (pepstatin), Cys-proteases (E64), trypsin-like proteases (TLCK) and chymotrypsin-like proteases (TPCK, 6-aminocaproic acid). Industry also offers inhibitor cocktails that handle at least standard situations.

Phenylmethylsulfonyl fluoride (PMSF) is an inactivator of serine proteases. It chemically modifies the thiol group in the active centre, so the proteases are send to kingdom come.

Benzamidine is a competitive inhibitor of serine proteases. In theory, it should not be required after PMSF, but only in theory  is there no difference between theory and practice.

So, in short, removing these substances from your homogenization buffer will not per se degrade your protein, but will give proteases (which are present everywhere) the chance to do so. 

Your yield may increase further if you add inhibitors or inactivators of metallo-proteases (EGTA, EDTA), Asp-proteases (pepstatin), Cys-proteases (E64), trypsin-like proteases (TLCK) and chymotrypsin-like proteases (TPCK, 6-aminocaproic acid). Industry also offers inhibitor cocktails that handle at least standard situations.

Hi there,

The behavior of your protein will also depend on its intrinsic susceptibility to serine proteases. For instance a very compact protein structure will be less sensitive than a partially disordered protein structure because accessibility to cleavage site will be hampered by the increased level of structuration.

Adding PMSF will not be having much adverse effect on your protein of interest unless it itself is a susciptible serine protease. In fact for all other protein types it would help by removing those pesky proteases that might just chew up your protein. Further, PMSF has a half life in the order of 102 minutes in aqueous solutions [it will be lesser if pH is in the alkaline range than in neutral zone at 25 degree centigrade]. Adding a cocktail of protease inhibitors will actually help you out in most cases.

Reference regarding half life of PMSF:

http://www.sciencedirect.com/science/article/pii/0003269778907844

If you use PMSF to inhibit proteases that might degrade the protein that you are interested in studying, preparing a concentrated stock solution in 2-propanol, and adding it to your buffer just before using it will help cope with its short half-life. If the organic solvent is incompatible with your experiment, AEBSF can be used instead. Both PMSF and AEBSF can also inhibit cysteine proteases by reacting with the sulfhydryl group at the active site. Inhibition of the cysteine proteases by PMSF or AEBSF can be reversed by treatment with a sulfhydryl reagent such as DTT, but inhibition of serine proteases is irreversible. Another thing to keep in mind is that not all cysteine proteases are inhibited by E-64. One example are the C13 cysteine proteases that are not inhibited by E-64 but are inhibited by iodoacetate, iodoacetamide and/or mercurial reagents.