Jacob,
You can try several things, including the stuff already mentioned by others - EDTA, salt, etc. A very useful option to keep in mind is to check that excess imidazole isn't causing the problem. You can find out by simply diluting the fractions down, or by changing the resin. His-SELECT and Co-Talon are two resins that elute in reduced amounts of imidazole - for example, HisSELECT is washed typically with 10 mM and eluted anywhere from 10 to 200, usually around 50 mM. In several cases that I personally worked on, switching from Ni-NTA to one of these resins eliminated all the issues. Finally, if for some reason Ni-NTA has won your heart and you'd rather not abandon it - you can always put a desalting column in line with the IMAC, this way the protein would come out and get instantaneously exchanged into the buffer of your choice. Happy purifying, Artem _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Wong Sent: Thursday, February 14, 2008 7:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] rescuing crashing-out protein eluted from Nickel column Dear all, I just ran into this problem and would like to see if I could get some helpful tips before my protein completely crashes out. I have a protein as 6His fusion and it remained bound to the Ni resin with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM (added to 1XPBS). The protein seemed to be highly concentrated in the elution and began to get cloudy right away, with more and more precipitation produced over a matter of minutes. I felt so helpless, didn't know what to do, and then decided to add 5% of glycerol into one of the fractions but that made it even more cloudy (ohh no...). While the protein is dying in the tube, do you have some quick remedy for me? Thanks very much, -J.J.
