problem with solubilizing protein in formic acid - (Oct/30/2008 )
I'm having problem to solublize my proteins in 70% formic acid for cyanogen bromide cleavage. I've also tried higher percentage and TFA with no success. After formic acid is added to the protein, the mixture is cloudy and very viscous.
This is the basic protocol I'm using to express and purify my protein
- the protein is expressed as a fusion with KSI (ketosteroid isomerase) into the inclusion body in E coli
- the inclusion body is solubilized in 6M Guanidine-HCl and put through a nickle column
- the column purified protein (theoretically, should be mostly my fusion protein) is dialyzed to get rid of Guanidine and then lyophilized
- the dry protein powder should then be dissolved in formic acid and mixed with cyanogen bromide for cleavage reaction, but I can't get this step to work!
I have expressed other proteins with the same procedure, and they dissolve completely in formic acid. The only major difference is my current protein has more cysteines (5), but I don't see how that would affect the solubility.
Any suggestions as to why I would get a cloudy and viscous solution or how to properly solubilized my protien???
Thank you very much in advance for your responses!
can u add surfactant ie triton or tween? or salt?