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Bradford Protein Assay - Problem in BSA Standard Curve - (Aug/07/2012 )

I am trying to determine protein concentration but my standard curve does not look like a standard curve at all.
I used to use BSA stock prepared by distilled water but I got high protein concentration all the time.
Then my advisor suggested me to use RIPA instead of water. So I prepared a BSA stock prepared by 0.5mg BSA powder and 10 ml RIPA lysis buffer (10mg BSA powder/200 ml RIPA). I put 25ul, 50 ul, 75 ul of my stock and so on into each well. Then I used PBS for both my standard curve samples and unknown samples to complete 200ul (i am using 96-well plate). But this time detergent and Bradford Assay reagent precipitated.
I have to find a solution on my own. If anyone can suggest me a solution I will be very happy. I hope I can explain the steps I have done until now clearly.
Thanks in advance


we make our stocks and dilutions with water. then we prepare subtractive blanks with the buffer(s) in which our samples reside.


Check your bradford solutions haven't gone off, they are relatively unstable so may well have precipitated.