Hi There is a nice article by PSI in Nature Methods (http://www.nature.com/nmeth/journal/v5/n2/abs/nmeth.f.202.html) about the generic buffers for Ni column. The article or the magazine issue is a very good reference even if you don't like those throughput guys.
One minor detail, the imidazole stock needs to be pH adjusted to the same pH as the buffer. No matter how strong a buffer is, at the concentration (25-100 mM) most people use, one should not expect a buffer can buffer imidazole in the Ni elution buffer, since imidazole is a very strong buffer itself. 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 [EMAIL PROTECTED] www.accelagen.com -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Valerie Guillet Sent: Friday, February 15, 2008 9:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column Hi, I also encounterd the same kind of problem with my protein. I noticed that the protein eluted from Ni-NTA with 150mM imidazole or 300mM didn't behave in the same manner; ie on gel filtration experiments, the 300mM eluate totally came in the excluded volume while the 150mM eluate came in the expected elution volume for a monomeric protein (at least for a few hours). Finally, the total protein was aggregated the day after. Using DLS experiments (dynamic light scattering), it was shown that the imidazole induced the aggregation of a monomeric sample. No reversibility was observed whatever I added in the aggregated form. Finally, I chose to elute my protein with a pH gradient with Na acetate buffered from pH 6 to 4 but without any imidazole. This new protein is really less sensitive to aggregation (but much more to proteolysis !!! With the protein eluted from imidazole gradient, the protein was stable on months but was completely aggregated!). Good luck Valerie Artem Evdokimov a écrit : > > 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 isnt 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 youd 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. >
