JJ
Dilute your fractions for this prep. Do gradient elution in the future. Again dilute the fractions immediately. Figure out a better buffer will be helpful. Cheers, Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 <mailto:[EMAIL PROTECTED]> [EMAIL PROTECTED] www.accelagen.com <http://www.accelagen.com/> _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Wong Sent: Thursday, February 14, 2008 4:26 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.
