I performed protein extraction from cell pellets using RIPA1X buffer and ROCHE's protease inhibitors cocktail cOmplete. When take it to Bradford protein quantification (Bio-rad) protocol, I get all my standards and samples absorbance readings at 595nm over 1.000 , even in the blank with only RIPA1X buffer and no proteins. Today I ran a test with RIPA buffer 1x and 0,1x with and without inhibitors with no mayor differences at the same concentrations, discarding the inhibitors as the problem source.
Results:
1x w/o inhibitors: 1.104
0,1x w/o inhibitors: 0.451
1x w/ inhibitors: 1.008
0,1x w/ inhibitors: 0.438
dH2O: 0.397
Run a standard curve with BSA and 0,1xr RIPA buffer getting a curve with r2=0,89 but weird as if it was kind of "saturated."
What can be the problem? I already checked all the variables and have no answer yet.
EDIT.
RIPA Lysis Buffer :
1% NP-40
0.1% SDS
50 mM Tris-HCl pH 7.4
150 mM NaCl
0.5% Sodium Deoxycholate
1 mM EDTA