Hi Fred, I am not sure if this has been brought up already. Another possibility might be to generously pipet your Ni- or Co-matrix of choice directly to buffered solubilized inclusion bodies, incubate for some time (even overnight if you feel its necessary), and then use the slurry to pack a column for further chromatographic manipulations. To quickly check if this approach might be promising, you can do this on a small scale in epps. IN fact working on a small scale like this might allow you to test a number of buffer choices and incubation protocols. You can spin down your beads followed by several washing steps with your column buffer. You can then go two ways: (1) either pellet with a hard spin and just add your SDS-PAGE sample buffer to the pellet to visualize binding on gel (make sure you boil your sample) OR (2) 'elute' your protein with your imidazole-containing buffer followed by analysis of the supernatant via SDS-PAGE. We have found that this approach works quite well for difficult metal-affinity chromatography cases.
Best wishes Savvas -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred Sent: Wednesday, January 28, 2009 9:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind Just to let you know. No way, Talon also don't work. I am gonna try the GE His-trap column. Fred wrote: > Hi everyone, > Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). > I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. > I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. > All the Best, > Fred >> >> >> --- Fred /<ccp4bb.l...@gmail.com>/ schrieb am *Di, 27.1.2009: >> * >> >> *Von: Fred <ccp4bb.l...@gmail.com> >> Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind >> An: CCP4BB@JISCMAIL.AC.UK >> Datum: Dienstag, 27. Januar 2009, 22:00 >> >> * >> >> *Hi ccp4 list, >> I am trying to purify a his-tag protein by metal affinity chromatography. The >> protein was expressed in inclusion bodies and its his-tag doesn't bind the >> Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with >> NaCl and detergents didn't help much. >> Any help is appreciated. >> Fred * >> >> E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11640 http://www.pctools.com/en/spyware-doctor-antivirus/
