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
[ccp4bb] AW: [ccp4bb] Off topic: His-tag purification
Hi Theresa, did you try to change the position of the His-Tag? Perhaps it is not exposed in your current construct and would work better at the N- or C-terminus. Greetings Moritz Metlitzky, Ph.D. Institute of Toxicology Tel.: 089-3187-2669 Fax: 089-3187-3449 www.helmholtz-muenchen.de/en/toxi/krappmannhttp://www.helmholtz-muenchen.de/en/toxi/krappmann 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 Helmholtz Zentrum M?nchen Deutsches Forschungszentrum f?r Gesundheit und Umwelt (GmbH) Ingolst?dter Landstr. 1 85764 Neuherberg www.helmholtz-muenchen.de Aufsichtsratsvorsitzende: MinDir?in B?rbel Brumme-Bothe Gesch?ftsf?hrer: Prof. Dr. G?nther Wess und Dr. Nikolaus Blum Registergericht: Amtsgericht M?nchen HRB 6466 USt-IdNr: DE 129521671
[ccp4bb] La Caixa/IRB Barcelona PhD fellowships Programme selection procedure
Dear colleagues, Please forward this information to students in your institutes who might be interested. L http://www.irbbarcelona.org/files/File/call2012lacaixa.pdf a Caixa/IRB Barcelona International PhD Fellowship Programme Structural and computational biologists IRB Barcelona is an independent, non-profit research institution engaged in basic and applied biomedical sciences, located at the Parc Científic de Barcelona on the University of Barcelona campus. We provide students in the life sciences from across the world with the opportunity to do training and research toward their degree in a unique international and multidisciplinary scientific environment. · A total of 10 doctoral fellowships are available for the academic year 2012-2013, under the auspices of la Caixa, the third largest Spanish financial institution · Open to students in the last year of their undergraduate studies, too. · Deadline for applications: 24th January, 2012 More information and application forms can be found at: http://www.irbbarcelona.org/index.php/en/training-and-jobs/phd-fellowships/ la-caixairb-barcelona-international-phd-programme-fellowships http://www.irbbarcelona.org/index.php/en/training-and-jobs/phd-fellowships/l a-caixairb-barcelona-international-phd-programme-fellowships Best regards, Esther Fernández Secretary Structural Computational Biology Programme Institute for Research in Biomedicine (IRB Barcelona) Parc Científic de Barcelona C/ Baldiri Reixac 10, 08028 Barcelona - Spain Tel. +34 93 403 99 53http://www.irbbarcelona.org/ www.irbbarcelona.org
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
[ccp4bb] Postdoctoral Position in Mechanistic Enzymology at the University of Iowa
Postdoctoral Position in Mechanistic Enzymology at the University of Iowa The Dey lab in the Department of Chemistry at the University of Iowa seeks to recruit an outstanding postdoctoral scientist with experience and/or interest in mechanistic enzymology. The successful candidate will be responsible for the characterization of enzymes involved in one of the research areas of the lab: methanogenesis; sulfur metabolism; mammalian oxygen sensing. The lab uses a combination of biochemical, structural, and spectroscopic techniques to investigate enzyme catalysis. Previous experience in structural biochemistry and enzymology would be valuable. This position provides the opportunity for broad training in protein expression, crystallization, kinetics, spectroscopy, genetics, and molecular biology. Candidates must have their Ph.D. by the beginning date of the appointment in Biological Chemistry or a related discipline (Chemistry, Biochemistry, Molecular Biology and Biophysics, Microbiology, Pharmaceutical Sciences, Biochemical, and Biomedical engineering). The successful candidate will have strong experience in molecular biology, protein expression, purification, crystallization, and functional analysis. A significant publication record is an advantage. The candidate must have the ability to work independently and in a team. For a complete job description and to apply go to http://jobs.uiowa.eduhttp://jobs.uiowa.edu/ and apply to requisition #1717. The University of Iowa is an equal opportunity affirmative action employer. Women and minorities are encouraged to apply.
[ccp4bb] postdoctoral position at the MRC LMB Cambridge
A post-doctoral position in AMPA glutamate receptor biology (AMPA-Rs) is available in Ingo Greger's lab at the MRC LMB, Cambridge. This post is funded by MRC-Technology and aims to develop AMPA-R modulators targeting the receptor's N-terminal domain (NTD). We seek an experienced crystallographer with an interest in ion channels. This is a multidisciplinary, collaborative project involving ligand-observed NMR screens, biophysical approaches, ligand evolution and AMPAR electrophysiology. Sukumaran et al. 2011. EMBO J. 30, 972-82 Jensen et al. 2011. J. Mol. Biol. 414, 96-105 (Rossmann et al. 2011. EMBO J. 30, 959-71) please apply online via: http://www.jobs.ac.uk/job/ADT692/investigator-scientist/
[ccp4bb] Off-topic: ELNs
Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! Thanks, Seiji
Re: [ccp4bb] Off-topic: ELNs
Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! For an out of the box solution, you may want to take a look at Labguru ( http://www.labguru.com ). If you are looking for a 'free' alternative would consider using google sites ( https://sites.google.com/ ) to roll your own solution. I hope that helps. Take Care, Sean Seaver P212121 http://store.p212121.com/
Re: [ccp4bb] Off-topic: ELNs
My lab uses http://biochemlabsolutions.com/ELN/ELN.html Best, Alexei. On 1/16/2012 5:03 PM, Sean Seaver wrote: Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! For an out of the box solution, you may want to take a look at Labguru ( http://www.labguru.com ). If you are looking for a 'free' alternative would consider using google sites ( https://sites.google.com/ ) to roll your own solution. I hope that helps. Take Care, Sean Seaver P212121 http://store.p212121.com/
Re: [ccp4bb] Off-topic: ELNs
Hi Seiji, A nice web base solution is the iLabber from Contur. It has been a popular option for many academic labs around the globe. http://www.contur.com/home/ Cheers, Francisco Sr. Product Manager Accelrys, Inc. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Seiji Sugiman-Marangos Sent: Monday, January 16, 2012 1:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic: ELNs Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! Thanks, Seiji
Re: [ccp4bb] Off-topic: ELNs
Hi Seiji, I would recommend Labguru (www.labguru.com) which is cloud-based ELN and laboratory management solution. This tool encompass the management of experiment and projects, connects between experiments and individual samples, manages the different storage facilities down to the single tube and inter-connects the knowledge base of your labs (protocol, articles) to every experiment/project. There are both free accounts (for individual students in the lab) or paid accounts for whole lab solutions, which take use of labguru's sharing capabilities among users. The system comes with a fast and reliable human support. Take care, Chen --- Chen Guttman The Zarivach laboratory for Macromolecular Crystallography Building 39, Room 009B Ben-Gurion University of the Negev POBox 653 Zip Code 84105 Beer-Sheva Israel http://lifeserv.bgu.ac.il/wb/zarivach Tel. +972-8-6479519 Fax. +972-8-6472970 On Mon, Jan 16, 2012 at 23:28, Seiji Sugiman-Marangos sugim...@mcmaster.cawrote: Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! Thanks, Seiji
