Hello,
I have some problem when using RIPA buffer during reading BCA assay. I'm getting high value of the RIPA's absorbance so I'm getting negative values.
I have Standard curve diluted with PBS and 100ul samples with 200ul of RIPA(X2) and proteinase inhibitor (0.5ul). Then taken the 25ul for the assay plate+200ul WR (reagent A+B).
What is the right blanks for me? (should I substract PBS value from the standard curve absorbance and substract RIPA+PBS+PI from the unknown samples absorbance value?)
Maybe:
Blank 1 - PBS (for standard curve)
Blank 2 - 100ul PBS (instead the sample) + 200ul RIPA(X2) + 0.5PI (for unknown samples)
What can be the problem? and do you have some suggestions how to calculate it?
Also what is the right graph type? 4PL/point to point/linear regression?
Thank you!
The BCA assay is not compatible with reducing agents. If there is dithiothreitol, mercaptoethanol, or other reducing agent in your samples, it will interfere. See the product literature for a compatibility table.
The blank should ideally be of the same exact composition as the sample, so it should include RIPA(X2) and protease inhibitors in the same proportion as the samples.
I totally agree with Adam answer, and maybe would also include the same proportion of all substances of the sample buffer into the standard series.
Best,
Murat
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