I havee trying to extract and purofy proteins starting from fresh plant material but always getting a lot of oxidations and polyphenols sticked to my proteins. So i would like please to ask if you have any suggestion or protocol to overcome this problem, where it is blodking my experimwnts progress. Thanks for your comprehension. Regards.
Precipitate proteins by adding methanol containing 0.1% formic acid. After centrifugation, the supernatant should contain most of the non-covalently bound polyphenols. It is very difficult to remove covalently bound polyphenols (you may have to use some reducing agents, but it might affect your protein quality).
Mahesha M. Poojary Okay thanks, but just a question my protein pellet will be solubilized in Tris-HCl buffer, pH 7.4? and do you recommend methanol precipitation over 90 % ethanol precipitation or Ammounium sulfate precipitation?. regards.
Mahesha M. Poojary at what concentration methanol is for precipitation ? this may lead to loss of proteins or no ?
I would go for methanol precipitation simply because, most of the polyphenols are soluble in methanol. Meantime, it is a good solvent to precipitate proteins. You may go for 1:5 (sample:methanol) or 1:10 for precipitation. I do not know if your pellet is soluble at pH 7.4-it depends on the pI of your protein. You may increase or decrease the pH based on the solubility.
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