Hello fellow scientists, here i have a bit of a seemingly demoralizing issue with protein purification. Could you please help? I have attached a commassie image for clarity.
10mM imidazole, 50 mM NaH2PO4, 300 mM NaCl - Binding buffer
20mM imidazole, 50 mM NaH2PO4, 300 mM NaCl - Washing buffer
washing twice. Can i increase the number of washes to may be four or five to remove contaminating proteins? or does extensive washing decrease the contaminating proteins?
500mM imidazole, 50 mM NaH2PO4, 300 mM NaCl - Binding buffer
But it seems the target protein doesn't bind to Ni-NTA columns (Qiagen spin column).
Stratregy:
Reduce the concentration of the imidazole to below 5 mM or less in the binding buffer so as to facilitate binding but i will have an incredibly high amount of contaminating proteins. Then, do extensive washing hoping to remove the co-eluating proteins.
What do you think?
Are you simply dissolving NaH2PO4 to prepare the buffer? At that pH the His residues of the tag will be protonated, and the protein will not bind to Ni-NTA. You need to prepare phosphate buffer at least at pH 7.5 (8, preferably).
Hope it helps!
I adjusted the PH to 8 as recommended in the Qiagen expressionist manual. I have two versions (truncated forms of the protein and i noticed both don't seem to bind to the column well
Try performing the purification in denaturing buffer (e.g. including 8 M urea or 6 M GuHCl). If that works, then the protein is either interacting with the tag or sterically hindering the tag from binding the resin. You have two options there, either you do the purification under denaturing conditions and then refold the protein later (you could even refold while having the protein still bound to the column) or you can change the tag to a different location on the protein.
Hope it helps!
How do i refold the protein the protein while the protein is still bound to the column?
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