I have been resuspending protein isolated by Trizol reagent in 1% SDS and storing at -20 degrees C. But everytime I need to use the protein for quantification, electrophoresis, etc., I need to heat it to 50-60 degrees C for 20-30 minutes, for the protein to dissolve. It again reprecipitates at room temperature. I'm afraid it would degrade upon repeated freezing, thawing and heating.
Moreover, during protein quantification by Bradford reagent (Bio-Rad protein assay), the reaction solution turns green instead of blue, and even the control with just SDS added without the protein, gives OD comparable to that of the protein samples. But the reagent manual says it is compatible with 0.1%SDS (final concentration).
Does anyone have suggestions for a resuspension buffer that would not keep throwing protein out of solution, and not also interfere with the quantification assays?
For resuspending a protein pellet after Trizol, we use 8M urea with some detergent as well (0.1% SDS or SDC) in a buffer. But! do not heat samples with urea, keep it under 25°C, carbamylation of proteins occurs. It might be a problem for consecutive proteomic analysis. We just shake it for 30 minutes as Julian suggested.
After dilution (10x) the Bradford assay is no longer a problem.
Thanks Julian and Jan.
@Julian, switching to Lowry's method is an option I can try out.
@Jan, I could try diluting the protein before quantification. Which protein do you use for standard curve? And do you use the same buffer (8M Urea & 0.1% SDS) for the standard curve as well? I had used 0.02% SDS in the final Bradford reaction, and I still got green colour in the blank and protein samples, which absorbed almost equally at 595nm.
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