[ccp4bb] crystallization of complex and ...
Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
Re: [ccp4bb] crystallization of complex and ...
Dear Min, Regarding the stoichiometry that you should use in crystallizing two proteins that form a complex. I have looked at this question before. See: Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G., Silverman, G.J., Charbonnier, J.-B. (2001) Crystallization of macromolecular complexes: Stoichiometric variation screening. J. Cryst. Growth 232:580-590. http://www.sciencedirect.com/science/article/pii/S0022024801011721 Briefly: The stoichiometry can, and should, be varied in your screening. The relative protein solubility of the two individual proteins and the solubility of the complex should be analysed under various potential crystallization conditions. Conditions where the complex is less soluble than the individual ptoteins should be chosen if the complex has a tendency to dissociate. Since both proteins have been crystallized before you may also use crystals of both the free forms of the two proteins to stimulate nucleation of the complex The final composition of the asymmetric unit may include free poteins as well as the complex. The paper will give you a lot more methodology to use to obtain crystals of the complex, if such complex really exists, is homegeneous and relatively stable. Enrico. On Fri, 16 Sep 2011 04:20:26 +0200, m zhang wrote: Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] crystallization of complex and ...
On Thu, 2011-09-15 at 22:20 -0400, m zhang wrote: > Second is about reusing of Ni-NTA resin. According to Qiagen's > instruction, after using fresh Ni-NTA resin, one only needs to wash > the used Ni resin first with 0.5M NaOH, then with your own buffer. > After that the resin is ready to be reused until it needs being > recharged. But my question is: Once immidazole competes with > His-tagged protein and binds to Ni-resin, how can immidazole be rinsed > off with the same buffer(usually pH is above 7) one uses to purify the > protein? Imidazole binds much weaker than a his-tag, and thus more of it goes into the buffer when you wash the column. In theory, if you wash a column with protein bound long enough, it will all slowly come off. -- "Hurry up before we all come back to our senses!" Julian, King of Lemurs
Re: [ccp4bb] crystallization of complex and ...
1 imidazole affinity is not high which is why you use 200 mM or more to elute. So it comes off by itself. 2 you can wash with low ph and then recharge this is somewhat easier on the resin On Sep 15, 2011 9:25 PM, "m zhang" wrote: > > > > > > Dear all, > I have two questions: > First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? > Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? > Thank you for any suggestion or comment. > Min >
[ccp4bb] AW: [ccp4bb] crystallization of complex and ...
Dear Min, regarding #1 some things come to my mind: - What is the Kd that you got from the Biacore? And did you make sure that the sample is concentrated enough (both on gel filtration and in crystallization) to have a "sufficient" amount in the complex. - did you use the same buffer systems? Your Kd might be different in different buffers. Best Alex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH & Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com <mailto:alexander.paut...@boehringer-ingelheim.com> Boehringer Ingelheim Pharma GmbH & Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Mark Hagmann, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von m zhang Gesendet: Freitag, 16. September 2011 04:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] crystallization of complex and ... Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
Re: [ccp4bb] AW: [ccp4bb] crystallization of complex and ...
To all and Alex, -The Kd is around 500nM from Biacore. -One of my protein tends to precipitate and is only stable below 3mg/m. If I concentrated it to higher concentration, it will precipitate a lot after 1hr. And it was previously crystallized at low concentration. But when I inject on gel filtration, I do try to load large amount both proteins. Then this leads to another question: How high should the concentration of complex be in co-crystallization or gel filtration? I set mine at 6mg/ml for the complex and I get some precipitate. What concentration of complex would people start with to co-crystallize? -Yes, I use the same buffer system for both proteins and complex. Thank you all for your suggestions. Min Date: Fri, 16 Sep 2011 09:13:46 +0200 From: alexander.paut...@boehringer-ingelheim.com Subject: [ccp4bb] AW: [ccp4bb] crystallization of complex and ... To: CCP4BB@JISCMAIL.AC.UK Dear Min,regarding #1 some things come to my mind:- What is the Kd that you got from the Biacore? And did you make sure that the sample is concentrated enough (both on gel filtration and in crystallization) to have a “sufficient” amount in the complex.- did you use the same buffer systems? Your Kd might be different in different buffers.BestAlex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH & Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH & Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ralf Gorniak, Mark Hagmann, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited.Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von m zhang Gesendet: Freitag, 16. September 2011 04:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] crystallization of complex and ... Dear all, I have two questions: First, I was trying to crystallize a complex of two proteins. Both proteins has been crystallized before. The two proteins bind to each other based on Biacore study, but they didn't form a single peak on gel filtration. When I mixed them at 1:1 ratio, the crystals I got contain only one of the two proteins. I was suggested to increase the ratio, for example 1.5:1, to increase the probability of co-crystallization which I will try. But I do want to hear if there are other possible ways to try. What would you try if you were in my situation? Second is about reusing of Ni-NTA resin. According to Qiagen's instruction, after using fresh Ni-NTA resin, one only needs to wash the used Ni resin first with 0.5M NaOH, then with your own buffer. After that the resin is ready to be reused until it needs being recharged. But my question is: Once immidazole competes with His-tagged protein and binds to Ni-resin, how can immidazole be rinsed off with the same buffer(usually pH is above 7) one uses to purify the protein? Thank you for any suggestion or comment. Min
