Re: [ccp4bb] Off topic: His-tag purification
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Theresa, you might also try changing the resin. I have worked on a protein which would no bind to Ni-NTA (Qiagen) at all but could greatly be purified when using Ni-IDA (then Pharmacia) instead. The protein expressed to about 60mg/l LB in E. coli and was well folded. Best wishes, Tim On 01/15/2012 07:23 PM, Theresa H. Hsu wrote: Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPFASdUxlJ7aRr7hoRAgiUAKD6Rzwp4nEtT4R/2xycc+k4Z0oKqgCg1HOn HgglN/BkelWqK8AFbn+IYHg= =HrLG -END PGP SIGNATURE-
Re: [ccp4bb] Off topic: His-tag purification
Hi Theresa, You could try lowering the pH down to 7.4 or 7. I have found that some proteins bind very poorly at pH8 and higher. Hope this helps. Cheers! --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: Theresa H. Hsu theresah...@live.com To: CCP4BB@JISCMAIL.AC.UK Sent: Sunday, January 15, 2012 1:23:33 PM Subject: [ccp4bb] Off topic: His-tag purification Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
…alternatively, depending on what is the source of your protein, you might have to dialyze the (concentrated) sample before applying it to IMAC. Insect cell media, in particular, can be pretty good at stripping off Ni2+ from IMAC supports. HTH, Luca
Re: [ccp4bb] Off topic: His-tag purification
Maybe the His Tag is blocked by the folded protein. Try using 6M Guanidine-HCL and see if it sticks. Then you will need to find some way of refolding your protein, if you want to crystallize it. Theresa H. Hsu wrote: Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
Your protein is either misfolded/aggregated or the his tag is chewed off/never translated. You could try detergents etc. To improve the state of the protein but I would also doubl check sequence for early termination (assuming cterm) or protelysis. Artem On Jan 15, 2012 12:23 PM, Theresa H. Hsu theresah...@live.com wrote: Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
I addition to the suggestions of checking folding/aggregation/proteolysis, you might also want to try lowering the NaCl concentration. Whereas I like having some to prevent ion exchange effects on the IMAC column, I have had the case of a protein that does not bind if NaCl is present. Binds perfectly well without, is eluted fine with 200 mM Imidazol. This protein does not bind at low Imidazol concentration either by the way. hth, Cécile Le 15/01/12 19:23, Theresa H. Hsu a écrit : Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa -- Cécile Breyton Institut de Biologie Structurale UMR 5075 CNRS/CEA/UJF 41, rue Jules Horowitz 38027 Grenoble cedex 1 France --- Tel: +33 (0)4 38 78 30 37 Fax: +33 (0)4 38 78 54 94 Courriel : cecile.brey...@ibs.fr http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109
Re: [ccp4bb] Off topic: His-tag purification
This likely means that your imac column is acting as an ion exchanger :-) On Jan 15, 2012 2:50 PM, Cécile Breyton cecile.brey...@ibs.fr wrote: I addition to the suggestions of checking folding/aggregation/**proteolysis, you might also want to try lowering the NaCl concentration. Whereas I like having some to prevent ion exchange effects on the IMAC column, I have had the case of a protein that does not bind if NaCl is present. Binds perfectly well without, is eluted fine with 200 mM Imidazol. This protein does not bind at low Imidazol concentration either by the way. hth, Cécile Le 15/01/12 19:23, Theresa H. Hsu a écrit : Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa -- Cécile Breyton Institut de Biologie Structurale UMR 5075 CNRS/CEA/UJF 41, rue Jules Horowitz 38027 Grenoble cedex 1 France --- Tel: +33 (0)4 38 78 30 37 Fax: +33 (0)4 38 78 54 94 Courriel : cecile.brey...@ibs.fr http://www.ibs.fr/groups/**membrane-and-pathogens-group/** ssimpa/article/ssimpas-1109http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109
Re: [ccp4bb] Off topic: His-tag purification
Hi Theresa: If you can make sure that your target protein is expressed. You can first use 6 M urea to denature the protein and then try to bind it to the column. If the denatured protein can bind to the column, it seems the histag is hided inside of the protein. It is not exposed enough to interact with the column. In this case, you can design a new construct, for example, put the tag to the other end of the sequence, or introduce a flexible linker between the tag and the protein. Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Good luck Yu Xiaodi Date: Sun, 15 Jan 2012 18:23:33 + From: theresah...@live.com Subject: [ccp4bb] Off topic: His-tag purification To: CCP4BB@JISCMAIL.AC.UK Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
Hi Theresa, I am right in thinking the protein construct you are using is just 8 His residues added to 90kDa ? It could be (if this is the case) that the tag is not accessible for binding to the Ni column. Sometimes you need a linker sequence between the protein for the His-tag to coordinate well to the Ni2+. Hope that helps Cheers Jodie From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Theresa H. Hsu [theresah...@live.com] Sent: Monday, 16 January 2012 7:23 a.m. To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: His-tag purification Hi all I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding? Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0. Thank you. Theresa
Re: [ccp4bb] Off topic: His-tag purification
On Sun, 15 Jan 2012 23:12:46 +, Xiaodi Yu uppsala@hotmail.com wrote: Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Can I know what is difference in binding chemistry of Ni, Cu, Co and Fe? Is there specific rule for binding affinity versus purity? Good luck Yu Xiaodi Theresa
Re: [ccp4bb] Off topic: His-tag purification
Typically affinity goes down in order cu ni co zn mg Artem On Jan 15, 2012 7:35 PM, Theresa H. Hsu theresah...@live.com wrote: On Sun, 15 Jan 2012 23:12:46 +, Xiaodi Yu uppsala@hotmail.com wrote: Another thing you can try is using Cu ion instead of Ni ion. It will fasten the binding. Can I know what is difference in binding chemistry of Ni, Cu, Co and Fe? Is there specific rule for binding affinity versus purity? Good luck Yu Xiaodi Theresa
