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Bradford Protein Assay - Problem in BSA Standard Curve

Bradford Protein Assay

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#1 bamya

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Posted 07 August 2012 - 05:18 AM

Hi
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

#2 mdfenko

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Posted 07 August 2012 - 08:30 AM

we make our stocks and dilutions with water. then we prepare subtractive blanks with the buffer(s) in which our samples reside.
talent does what it can
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#3 bob1

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Posted 07 August 2012 - 02:07 PM

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





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