Hello everyone,
As I wanted to see an agent's activity on my cells, I have measured DNA fluorescence via Hoechst dye. This agent prevents chain elongation by incorporating into DNA, afterward inhibits DNA synthesis.
My question is if it is possible to perform another experiment on the same plates. I mean without dumping the remaining solutions, can we add Bradford and measure protein for example? If it is not possible, what another kind of experiments can I apply?
(The plates now contain "100 ul 1x SSC + %0.02 SDS" and another "100 ul 1x SSC containing 2 ug/ml Hoechst".)
Since some of my results are not as I expected, I want to validate them. Thank you for help in advance.
I think this would be a problem. For one thing, the SDS detergent will interfere with the Bradford assay. You might be able to perform the micro BCA or Lowry assay on the samples from the wells, although I suspect the protein concentration may be too low to measure.
Hello, what I cannot figure out is how you can add Bradford reagent directly to your cells in the plate? You don't need to harvest them first?
Dear Adam B Shapiro what if I put the same solutions also into the BSA standards? Can I eliminate the interference with this? I mean something like blank... Thank you for your suggestions.
Dear Iskander Madhi the cells are on the 96-well plates and because of the SDS they have already lysed. That is why I have thought that I can perform Bradford without doing an extra step. Thank you.
It might be OK. Here is a protein assay compatibility table, which indicates that some SDS is tolerable. Make sure that the standards contain the same composition as the samples.
https://assets.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0068-Protein-assay-compatibility.pdf
Best way to find it is by testing with standards of known protein conc. I always suspect which reagents interfere with Bradford's reagent. Also please include a buffer control. Good question.
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