Hello everyone,
I have been purifying protein A (pI =8.4) for crystallization. My problem is that this protein is precipitated during and after the concentracing process. Usually, the Tris-HCl buffer used in the concentracing process is: 0.15M NaCl, 20 mM Tris-HCl, 2% Glycerol, 5mM DTT . I also have tried to change some parameters like: increased glycerol up to 5%), pH 7 and pH 9... but it seems to be no useful... Please kindly let me know if you have any suggestions!
Thanks you
I would suggest increasing the salt concentration to 0.5M. That may shield electrostatic interactions between molecules. Also, consider that it may not be necessary to concentrate the protein so much.
Hi Dung, I am thinking of your protein concentration while reading your question. I usually obtain precipitated proteins when concentrating them too much. Hope this help!
Yes, it seems like concentration dependent precipitation but sometimes changing buffer also helps because different buffers have different pKa. In your case I think HEPES might help ? Good luck!
Thanks for your suggestions guys. @ Humanyun: I have tried already with HEPES buffer, that precipitation was still happens.
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