RIPA Buffer turns Bradford into Blue - (Mar/01/2011 )
I am using RIPA protein extraction protocol and in my first trial RIPA buffer in the blank of Bradford was blue, I got negative values with this blank. Later I tried each ingredient of RIPA buffer (SDS, NaCl, Tris, DOC, NP40, ddH2O)in Bradford one by one, only SDS and NP40 (2ul) turn Bradford into blue. I freshly used these 2 and eventhough SDS and NP40 turned Bradford by themselves into blue, buffer was ok when I used it in blank. So problem was neither SDS nor NP40 I thought but after a week I have faced with the same problem. Do I need prepare everything fresh each time. Any idea?
My understanding is that Bradford assay is NOT compatible with detergents (only compatible with low concentrations of SDS or Triton see link) and therefore not compatible with RIPA buffer.
I used to use Pierce's BCA kit which is optimised for higher concentration of detergents (see link)
Hope this helps
yeah it happened to me too, my reagent turned blue also. As long as I did relative comparison to just normalize my samples I didn't care about the exact concentration. Although Bradford is a quick method, it is not entirely precise. Some people add BSA to lysis buffer when drawing standard curve, but I never did. I just added BSA to PBS. Also, my friends used to tell me not to use protease inhibitors when doing Bradford, but how can you not use PI if you are extracting proteins?