Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 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 Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: leonard.cha...@manchester.ac.uk http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 *
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Two things to be mentioned. * IDA columns bear an overall negative charge. I expect this behavior holds true with NTA gels. Hence salt, (= 0.5 M NaCl) must be present in your adsoprtion buffer to quench possible repulsive electrostatic interactions. * You are dealing with protein adsoption by coordination bond formation to a metal-chelate. Coordination bond lentghs decrease (and binding improves) as ionic strength increases, so a 1-2 M salt concentration in you buffer may turn out to be appropriate. HTH, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr 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 *
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 *
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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/
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi Fred, I doubt His-trap column be any better. I never found much a difference between these resins or columns. If you want to try, use His-Trap HP, not His-Trap FF. Doing batch binding may help you figure out the problem. In some cases, protein binds better in batch mode. You can take beads out at time intervals up to overnight for binding analysis. I guess not working means the protein in the flow-through. Sometimes for native purification, proteins don't come out of a column. But never experienced anything like this under denatured conditions. Good luck, Chun -Original Message- 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 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 *
[ccp4bb] [OFF TOPIC] his-tag doesn't bind
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
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 27 Jan 2009, Fred wrote: 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
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
You can also try TALON cobalt affinity resin from Clontech ( http://www.clontech.com/products/detail.asp?product_id=10588tabno=2) or the high yield PrepEase resin from USB ( http://www.usbweb.com/category.asp?special=cat=234id=78806). On Tue, Jan 27, 2009 at 4:34 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: 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 -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 27 Jan 2009, Fred wrote: 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 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 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
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
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 xtald...@gmail.com wrote: 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 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 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
