Re: [ccp4bb] Concentrating purified membrane protein
Hi Raji, DDM has quite a large micelle so you might be alright with a 100kDa cut off concentrator, I've had the same experience with a 30kDa protein in LDAO being comfortably contained by a 100kDa concentrator. I would try with a small amount of you sample and see if significant amounts of protein are found in the flow through. Is your protein tagged? if so you could bind it to a small volume (1ml) affinity column, exchange it into the lowest DDM conc. it is stable in (0.01% ?) and elute with a very steep gradient. If this didn't give you the desired concentration at least it would minimize the volume to be concentrated (and thus the final detergent concentration). You could also try and intermediate cut off size concentrators you have available (20,30,50kDa etc) until you find biggest one that retain the sample. Good luck Rhys Grinter From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji Edayathumangalam [r...@brandeis.edu] Sent: 14 July 2013 01:47 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Concentrating purified membrane protein Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Concentrating purified membrane protein
Well, you'll never loose the protein in the flowthrough if you don't discard the flowthrough.which i never do until I'm sure about what happened to the protein. But yes, try different Mw cutoff concentrators until you find the highest one that works. Depending on the purification your sample may still contain lipids, which will make the micelle size even bigger compared to pure DDM. Also, some concentration of the detergent is not a problem. One tends to get more phase separation in crystallization trials with higher detergent concentrations but that does not necessarily prevent crystallization. If your protein is really stable and you're very worried about higher detergent concentrations, you can dialyze the protein against your buffer solution. Again try different cutoffs. You're not aiming for complete equilibration (which could take a long time), but to bring the concentration down via once or twice overnight dialysis. Personally we used to dialyze everything prior to freezing the protein but we have moved away from that w/o obvious effects on success rates. Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Sunday, July 14, 2013 1:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Concentrating purified membrane protein Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Open Postdoc position in X-FEL crystallography, Frankfurt/Germany
In a joint project of the Goethe University Frankfurt/Germany and the Max-Planck-Institute of Biophysics and funded by the Cluster of Excellence for Macromolecular Complexes, we have an open position for a Postdoc working on structural characterization of membrane protein complexes by X-ray free electron laser (X-FEL) crystallography. We are looking for a highly motivated person who is particularly interested in developing new approaches for crystallization and structural analysis of large membrane protein complexes. The candidate must have a strong background in structural biology and experience in membrane protein purification and crystallization. Experience in collection and processing of X-ray diffraction data as well as preferentially also in X-FEL crystallography is desirable. We offer a highly stimulating and well-equipped research environment, integrated in the life science campus Riedberg with its exceptional expertise and interest in macromolecular (membrane and soluble) complexes (http://www.cef-mc.de http://www.cef-mc.de/). The position is available from July 2013, and the initial appointment is for 1 year, with an option for extension. Consideration of applications starts when this job advertisement appears. This call closes on July 15, 2013. Please submit your application including CV and further information (list of publications, contact of three referees, etc.) per e-mail to: thomas.me...@biophys.mpg.de mailto:thomas.me...@biophys.mpg.de -- Dr. Thomas Meier Research group leader Max-Planck Institute of Biophysics Department of Structural Biology Max-von-Laue Str. 3 DE-60438 Frankfurt/Main Phone: +49(0)6963033038 www.biophys.mpg.de/meier
Re: [ccp4bb] Concentrating purified membrane protein
Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Concentrating purified membrane protein
can you elute off the column with low pH ? Or EDTA if you don't want imidazole around ? Jürgen On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote: Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.edumailto:r...@brandeis.edu wrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] Open Post-doc positions in Structural Immunology with a focus on enhanced TCR recognition of MHC molecules at the Science for Life Laboratory (scilifelab.se) in Stockholm, Sweden.
Open Post-doc positions in Structural Immunology with a focus on enhanced TCR recognition of MHC molecules at the Science for Life Laboratory (scilifelab.sehttp://scilifelab.se) in Stockholm, Sweden. The newly created SciLifeLab, which is a joint effort between four Swedish universities, including the Royal Institute of Technology (KTH), the Karolinska Institute (KI), Stockholm University (SU) and Uppsala University (UU), serves as a Swedish national infrastructure to support infrastructure and technically advanced research in the life science area. The infrastructure at SciLifeLab includes state-of-the-art technologies for high-throughput molecular biosciences. We are currently seeking for one or two highly motivated and multi-talented post-doctoral fellows to work on a collaborative project in the Achour laboratory within SciLifeLab. This is an ideal position for a post-doc who is looking to bolster experience and/or publication record for several years before moving to a faculty position. The selected post-doctoral fellow(s) will focus on determining the three-dimensional structures of T cell receptors in complex with MHC class I or class II, presenting wild-type and altered versions of epitopes related to autoimmune and/or cancer diseases. The overall aim of the project is to establish and understand the mechanisms underlying enhanced TCR recognition of infected and/or cancer cells, as well as to provide a structural basis for the induction of autoimmune responses. All the structural studies will be performed in parallel with functional assays within the research groups of Prof. Lars Klareskog and Dr Vivianne Malmström (both at the Center for Molecular Medicine (CMM, KI), as well as the research group of Dr Petter Brodin (SciLifeLab, KI). A successful PhD in a relevant scientific discipline and proficiency in structural and molecular biology are required. The post-doc fellows will have a lot of autonomy, but are also expected to provide theoretical and technical assistance with overall laboratory computational and crystallographic projects. The candidates should have a strong will to work on challenging and significant protein complexes in a multidisciplinary setting. Main skill sets required are facility in cloning and protein production, practical aspects of protein crystallization, structure determination and computation. Familiarity with protein expression systems, cell culture, various biochemical and biophysical techniques to study protein-protein interactions are also highly desirable. The candidates are expected to be particularly interested into determining the three-dimensional structures of protein complexes, as well as assess biophysically their interactions using e.g. Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC) or microscale thermophoresis. A large panel of other biophysical technologies is also available within SciLifeLab. The positions are initially offered for a period of two years (1+ 1year), but if successful, the post-doctoral fellows will also be offered a portfolio of their own projects in the areas of structure and/or protein engineering, as well as possibility to establish an own research group. The applicants should send a CV, with a complete list of publications (only published manuscripts) as well as a letter in which they describe the basis of their interest in the proposed project. Three professional references should also be sent in which the scientific and social qualities of the candidate are described. All required information should be sent by e-mail to adnane.ach...@ki.semailto:adnane.ach...@ki.se. More information about the Achour group can be found in scilifelab.sehttp://scilifelab.se as well as in cim/ki.se. The positions are available from the beginning of September 2013, and will remain open until filled. The initial appointment is for 1 year. Consideration of applications starts when this advertisement appears. This call closes on December 1th, 2013. Adnane Achour
Re: [ccp4bb] Concentrating purified membrane protein
Raji, One point the most people forget about is that whenever you concentrate any detergent-solubilized membrane protein is that you will ALWAYS concentrate the detergent. So regardless of the MWCO, if the protein-detergent complex concentrates, the overall detergent concentration also increases. What you want to shoot for is balancing protein loss with obtaining a sample having minimal EXCESS free detergent. While a concentration step with a Ni-column followed by dialysis will work, so will a wise choice of concentrator. One trick to moderate high excess detergent is just to avoid the need for a many fold concentration where you will really concentrate the free detergent. Nonetheless, we have used concentrators effectively, and a Ni-column followed by dialysis as well, but if you still have too much free detergent, you can always use a spin desalting column with G-25 sephedex to bring remove detergent down to a nominal concentration. In all cases, you will lose some protein. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote: Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
