I'm trying to perform a BCA assay on some clarified lysate, but the samples appear to become brown, not purple, and the OD562 absorbance is very high. What could cause this?
The brown is very distinct from the normal purple of the BSA standard.
The BSA standard has my same lysis buffer (2% SDS), so I don't think it's anything in my lysis buffer.
Do you have a reducing agent in the sample? The BCA assay is sensitive to them. Here is a compatibility table for various protein assays.
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
Gen Gi Have you made sure the buffer has a neutral pH? The brown colour would be a little bit strange for a BCA assay, however. In a Bradford assay, that colour would be way more common and would indicate no protein binding to the coomassie.
Hi Gen Gi,
Have you prepared the standard in the same buffer as used for the sample? Any difference in composition might have created the difference in colours.
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