I did my cell line assay in 96 well plate and wanted to do the corresponding protein assay in 6 well plate. For 96 well plate the seeding density i used was 40,000cells / well in 100ul final volume. I did the same experiment for immunoblotting in a 6 well plate. The cell seeding was 1.1 million cells/2ml [40,000/0.32cm2 X 9.5cm2= 1.187500]. But i couldn't get the same result with protein as expected. Is there something wrong?
Also can anyone suggest the calcium concentration appropriate for the protein assay? i need ca for the experiment to work. I used 10uM final concentration which is good. Does it affects the results?
Your math is correct, so problem lies somewhere else. If you use Bradford assay kit of some sort, 10mM Ca should not interfere with it - so it's OK to use 10mM Ca. Just make sure you use the same buffer in control wells, just in case. Here is a Sigma protocol that has compatibility table at the end - check if any other component of your buffer interferes withe readings:
http://www8.umoncton.ca/umcm-gauthier_didier/bc4993/instruc/bradford.pdf
Good luck!
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