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

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