Hi Ren, what alkaline proteinase are you using?

Steingrimur Stefanssonis right, it all depends on the protease family you're working with. Then you can find a specific inhibitor.

I have some experience with plasmin (a heat-stable alkaline protease of the serine proteases family) and in this case I would suggest to add PMSF or AEBSF to your medium, because these molecules are irreversible inhibitors of serine proteases (such as plasmin, trypsin, trhombin etc.)

My alkaline proteinase is an serine protease.As we known, Endotoxin detection usually used the LAL method which is enzymatic reaction, then if I add PMSF or AEBSF to my  medium it may be a interference to the  detection process

OK, now I see , you don't want to inactivate both the serine protease in your substrate AND the serine protease that starts LAL coagulation...

I don't know if this can help, but PMSF (not AESBF) is very unstable in water solutions (half life is measured in minutes, depending on pH and temperature) so I guess you could treat in advance your substrate with PMSF and it will form irreversible complexes with your unwanted protease. Then you can incubate for the right amount of time, to let excess PMSF be degraded and use your substrate in your LAL reaction. 

It would require some preliminary tests to understand the better combination of pH, temperature, time and PMSF concentration, but it seems to me that ic could work.

I'll browse for more information, now I'm curious... good luck!

Hi Ren, I think Leonardo has the simplest solution. PMSF has a half-life of ~30 min at pH 8.0

http://www.sciencedirect.com/science/article/pii/S0301562910006691

So you could incubate your proteinase with a 10x molar excess overnight at RT in  pH 8.0 buffer. As a control you could incubate the same concentration of PMSF in that buffer without the proteinase.

Thank you for both your suggestions.I found some protein precipitation when I added the PMSF(200mM/L ) to my protease .Protease that used for the test at a concentration of 3mg/ml in 50 mM Tris-HCL buffer pH 7.5.The PMSF:2ul、4ul、6ul、8ul、10ul was added to the 100ul protease respectively and protein precipitation appeared immediately.The least protein precipitation was the one that added 2ul PMSF.I considered the final concentration of PMSF may be too high，then the PMSF(200mM/L ) was diluted to 50mM/L with isopropyl alcohol.I made the same test .The protein precipitation appeared in the two test samples that added 8ul and 10ul PMSF.The two samples was centrifuged and the supernatant was used for enzyme activity detection. I found that the two samples still remained active and almost no difference with the original sample.I have no idea what is the reason

Mr Bianchi

    I would like to know your experience with plasmin because my protease is a kind of plasmin. Did you make the plasmin inactive completely with PMSF?

So if I understand weel, you first prepared a  200 mM PMSF solution in water and then diluted it 4 times (50 mM) in isopropanol? If it is so, I wonder if the precipitate you see could not be PMSF itself, which is hardly soluble in water.

I think that maybe you should first prepare a 100 or 200 mM solution directly in anhydrous ethanol, then you could try to add all the range between 1 µl and 0.1µl to your protease solution (final concentration 1-2 mM to 0.1-0.2 mM). Apparently solutions up to 5 mM concentration have been used to inhibit Proteinase K, I'd start with the smallest concentrations possible.

By the way, are you really testing a 3 mg/ml protease solution? isn't that a very high enzyme concentration? You could also try to dilute the substrate, or find the best conditions for your tests...

And yes, as little as 1 mM PMSF worked for plasmin in milk, and I see it is the higher concentration generally recommended. Unfortunately, PMSF is hard to solve in water, and it is instable, so Pefabloc is used instead. In your case, however, this instability is what you seek, so I'd give it some more try...

I first prepared the200 mM PMSF solution in isopropanol then used it for dilution.I donot know why the precipitation  appeared. May be  the enzyme concentration is too high and it  reacted with PMSF then precipitated .I will do more experiments to find the most appropriate  reaction conditions. 

Hi Ren I agree with Leonardo in that the precipitate you are seeing is probably the PMSF and not your proteinase.

But PMSF shouldn't precipitate immediately if the only salt in your preparation is 50 mM Tris-HCl.

Unless you made the buffer from Tris-HCl salt and added NaOH to bring it to pH 7.5.

Anyway, personally, I never used isopropanol to dissolve PMSF, I used DMSO. I found that DMSO is more forgiving when diluting hydrophobic molecules into aqueous buffers.