Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-29 Thread Chavas Leo
Dear Fred -- just to check... are you sure you have the His-tag? Might have been cleaved somehow? You might want to increase the number of His in the tag as well. HTH. -- Leo -- On 27 Jan 2009, at 21:00, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Chun Luo
ssage- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred Sent: Wednesday, January 28, 2009 12:08 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

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Savvas Savvides
y, 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 m

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Fred
DS-PAGE with samples eluted from Talon and let you know the results. > All the Best, > Fred >> >> >> --- Fred // schrieb am *Di, 27.1.2009: >> * >> >> *Von: Fred >> Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Nadir T. Mrabet
Fred 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

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-28 Thread Fred
27.1.2009: * *Von: Fred 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 express

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-27 Thread Jim Fairman
To go along with the EDTA comment, did you check the pH of your binding solution? Sometimes lysing E. coli causes the pH to drop below the pKa of the histidines. On Tue, Jan 27, 2009 at 8:10 PM, James Stroud wrote: > You don't have EDTA left over from your protease inhibitor, do you? Some > com

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-27 Thread James Stroud
You don't have EDTA left over from your protease inhibitor, do you? Some commercial cocktails include it. You may have to read the fine print. James On Jan 27, 2009, at 1:00 PM, Fred wrote: Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protei

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-27 Thread Jim Fairman
You can also try TALON cobalt affinity resin from Clontech ( http://www.clontech.com/products/detail.asp?product_id=10588&tabno=2) or the high yield PrepEase resin from USB ( http://www.usbweb.com/category.asp?special=&cat=234&id=78806). On Tue, Jan 27, 2009 at 4:34 PM, Tim Gruene wrote: > Hi Fr

Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-27 Thread Tim Gruene
Hi Fred, I used to worked with a protein which would not bind to the Qiagen NiNTA (really *NOT*) but beautifully to IDA resin (e.g. Amersham Pharmacia). This was under non-denaturing conditions, though, and I don't know whether this applies to the denatured state. It may be worth a try. Tim

[ccp4bb] [OFF TOPIC] his-tag doesn't bind

2009-01-27 Thread Fred
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