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protein loss during nickle clolumn purification - is there always a lot loss of protein during column purification? (Dec/29/2005 )

Hi, is there anybody experience such thing? When I do His-tagged protein purification with 1ml nickle column from Pharmacia, the collected target protein in the elution steps is only about one fifth of that in the originally loaded sample. Such a large amount loss!
I experienced similarly one year ago when I use 1ml ion exchange column(ironically, also from Pharmacia) to purify EGFP protein. I can never get enough protein for N terminal sequencing by ion exchange purification due to the huge protein loss.
Can anybody tell me why this happens? Is there always large amount protein loss when using such colums for purification? Of course I must say the target prtein in the oringinal sample is also not so much.


It's possible to saturate the column with protein and thus the remaining protein in solution will not bind. However it sounds like this is not the case here as you say there is not much protein to begin with. Do you know anything about the structure of your protein? Specifically, is the end with the his tag on it likely to be exposed to solution or buried within the protein? If the his tag is buried by the protein folding, it will not bind to the column. You may have better luck switching the tag to the opposite end of the protein (this has work many times for my colleagues). You may also try a denaturing purification to see if you then recover your protein. If you get much more protein recovered under denaturing conditions, it is likely that the his tag is buried and not accessible in the folded protein.


Thanks a lot.
Your first suggestion is right. It is because the low protein amount to begin with that makes me see the great loss. When I have large amount of samples, the problem is not a problem any more, at leat, I can not observe it.