A published method i'm trying to replicate calls for a RIPA buffer described as:

"(1 M NaCl, 0.5 M EDTA, 1% Triton X100, 0.5 M Tris-Cl pH 7.4; with added 5 M DDT, 0.1 M PMSF, 5 M mercaptoethanol, 3 mM protease inhibitor (PI) diluted 1:1000)"

I've had difficulty getting details from the author about other aspects of this work, and have given up trying direct communication. I have little experience with protein biology, but would like to reconstruct this method if possible...it would be a major step forward in our lab's work! My question for those with protein extraction experience is: how would you interpret the protease inhibitor part of the above recipe? To me, I think the most likely interpretation is:

1. make up the NaCl+TritonX+Tris buffer

2. make up the DTT+ PMSF + mercaptoethanol inhibitor mix

3. Dilute the inhibitor mix 1:1000 in the first buffer before using

However, the "3mM protease inhibitor" is confusing. Is "protease inhibitor "a reagent in its own right? From my reading, the DTT, PMSF and B-mercapto all act as protease inhibitors. Or perhaps 3mM refers to the concentration of a mistakenly omitted fourth inhibitor?

I just have no intuition for this kind of work, so i thought i'd ask the world. Any suggestions?