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protein in phosphate buffer precipitate out with acetonitrile - (Dec/21/2009 )

I want to quantify my protein by RP-HPLC. Usually my protein is just in water solution and I mix it with acetonitrile before loading onto the column. And it's fine. But recently my protein is in phosphate buffer. so when I mix my protein in phosphate buffer with acetonitrile, the protein would precipitate out. What should I do?

Thanks a lot!!


what is the pH of the phosphate buffer (sodium or potassium)? is it near the pI of the protein?

what is the concentration of acetonitrile? concentrated acetonitrile (ie high percentage or pure) can precipitate proteins.