I have been having an issue when coupling my GST/His-tagged protein to CnBr sepharose beads. When I add my protein to the coupling buffer and the CnBr beads, an egg-white-like precipitate forms after a few minutes of mixing (which I assume is the protein precipitating out!).
This is strange as the protein I am using has been dialysed in the same coupling buffer with which I use to couple the protein to the beads. I have already troubleshooted this extensively and it still can't explain this!
The first time I did this, the precipitation occurred but I carried on with the coupling and funnily enough it appeared to have worked as I was able to pull down proteins by western blot with the coupled beads!
Even so, I am still perplexed as to what could cause this and am worried if the efficiency of the coupling is being lost. I would appreciate any ideas!
(The first time I did this, I used 2.5mg protein with 0.5ml bed volume of CnBr sepharose beads. Since then, I scaled up using 5mg to 1ml bed volume beads).