I have expressed a viral protein in E.coli. and purified it using Ni affinity chromatography. I have refolded it in vitro and dialysed it into PBS. I then performed protein estimation by Lowry's method. While my standards are fine, I see a precipitate in my protein sample as soon as I add 1:1 diluted Folin's reagent. Initially I thought it is because of high concentration of protein in my sample. So I diluted the protein 1:1 and then used 5 and 10ul of the diluted sample for the estimation, I am still seeing the precipitate although it is slightly less.
Can anyone suggest a reason and a remedy for this?
Do you have detergents and/or reducing agents in the protein sample? That would explain the precipitation. BCA and Bradford are simple alternatives, all methods have pros and cons, you may want to try some or all of them depending on buffer composition. A280 is also a possibility (again, depending on composition of your sample).
In my expericen i have found that protein clustering happen before the cuantification method took place. I strongly recomend to use a densitometric approach for protein quantification or quantify after purification.
To avoid agragation you could try change the pH of the solution (diferent to isoelectric point) or add DTT
You probably don't have this problem anymore, but I had it and found a solution, so for the people that arrive here from Google search results... you can dissolve the precipitate by adding SDS. Article A simple technique for eliminating interference by detergent...
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