Generally I use RIPA buffer for protein lysis for western Blot from tissues and cells. But I have noticed it interferes while doing estimation of proteins by Bradford's method. However I also used STET buffer for the same and that too has same interference. May this be due to SDS. Is there any other lysis buffer which doesn't interfere during protein estimation and quantity of proteins is good?
All detergents, such as SDS, NP40, triton-X100,... interfere with quantification kit. In addition, your standard range is probably done with BSA, and the comportment of BSA with the quantification kit is not equal to the comportment of protein from cells.
To limit bias, I do my standard range in the lysis buffer. And I do all my quantification in the same buffer. In this case, I have a bias but it is the same for all samples.
Last, you could try a lowry's method to perform the quantification. I think that the change of the lysis buffer have too much impact on the solubility of your protein.
Hej Abhinav, I think doing the protein quantification with the BCA method instead of Bradford might also help, since detergents do not interfere with this method.
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