Re: [ccp4bb] protein purity in SAXS
Dear Li, In regards to concentration ideally you should do a concentration series and from that perform a zero concentration extrapolation. Typically we've had success with a 1-10 mg/ml series and 1.5-3 s exposures at SSRL but it's going to be dependent on molecular weight. You should monitor your signal to look for any up turn at low resolution that would indicate multi-particle interaction or aggregation. If you have signal in the lower concentration samples and you see this effect in the higher concentration ones then take the sequence to more dilute ones. Advice from the beamline scientist or instrument manufacturer should guide you on typical concentrations and exposure times for the source you plan to use. We found the review by Putnam, Hammel, Hura, and Tainer entitled "Solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution" (Q Rev Biophys. 2007 Aug;40(3):191-285) to be very useful for practical information. This is available at http://bl1231.als.lbl.gov/publications/index.php. There is also a good example of what to expect in your data at http://bl1231.als.lbl.gov/saxs_protocols/saxs_data_collection_evaluatio.php which is also in the paper. Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! > Dear Li, > > The SIBYLS Beamline web page has useful sample prep information at > http://bl1231.als.lbl.gov/saxs_protocols/index.php. They also have a link > to a very good SAXS review article. > > Cheers, > Angela > > > > > Li Sheng > Sent by: CCP4 bulletin board > 04/23/2009 06:19 AM > Please respond to > Li Sheng > > > To > CCP4BB@JISCMAIL.AC.UK > cc > > Subject > [ccp4bb] protein purity in SAXS > > > > > > > Dear all, > > Is there anyone with experience in small angle x-ray scattering? Could > anyone please tell me the requirements for protein sample purity and > concentration? > Thanx in advance. > > > > Sincerely, > Li > > __ > Email:s...@sibs.ac.cn > Institute of Biochemistry and Cell Biology > Institutes for Biological Sciences > Chinese Academy of Sciences > 320 Yue-Yang Road, Shanghai 200031, China > Tel: +86-21-5492-1217 > __ > >
Re: [ccp4bb] protein purity in SAXS
Since the SAXS signal comes from the size of your scattering particles, make sure you don't have large non-protein particles in your sample (dust or micelles, for example). The SIBYLS web site has additional warnings about including detergents in your sample. ho UC Berkeley
Re: [ccp4bb] protein purity in SAXS
Dear Li, The SIBYLS Beamline web page has useful sample prep information at http://bl1231.als.lbl.gov/saxs_protocols/index.php. They also have a link to a very good SAXS review article. Cheers, Angela Li Sheng Sent by: CCP4 bulletin board 04/23/2009 06:19 AM Please respond to Li Sheng To CCP4BB@JISCMAIL.AC.UK cc Subject [ccp4bb] protein purity in SAXS Dear all, Is there anyone with experience in small angle x-ray scattering? Could anyone please tell me the requirements for protein sample purity and concentration? Thanx in advance. Sincerely, Li __ Email:s...@sibs.ac.cn Institute of Biochemistry and Cell Biology Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road, Shanghai 200031, China Tel: +86-21-5492-1217 __
Re: [ccp4bb] protein purity in SAXS
Dear Li, as pointed out by Marjolein the sample must be MONODISPERSE, rather than just pure. This means it should be checked not only by SDS-PAGE but at least by size exclusion chromatography, and be sure that monodispersity is maintained till the moment of measurements. I guess your plans are to collect data at synchrotron even if you did not specify... In this case you have to check that some critical steps (like freezing the sample for shipping to the synchrotron) do not affect monodispersity. As a first check you might want to run a DLS analysis. This should be performed AFTER sample concentration (if possible up to 10 mg/ml for a 50-100kDa protein) to confirm that this step is not critical. Always remember to save the exact protein buffer for background data collection (flow through of the concentration device). An exact background subtraction is fundamental in order for your further data analysis to make any sense. Another step to check is the freeze-thaw procedure because aggregation might also happen at this stage... Try flash-freezing your concentrated protein in aliquots, then thaw one of them to see if DLS confirms that it is still monodisperse. If everything is ok you can repeat the procedure before measurements, otherwise you should find a way to avoid these problems. In some cases you might consider performing the final concentration step (or the entire protein purification) directly at the synchrotron immediately before measurements... or to ask for some advice to your local contact. Ensure also to have enough concentrated protein to perform one measurement at the highest concentration and 3-4 dilutions at lower concentrations. After measurements of the concentrated solution you cannot just recover it and dilute to measure another dataset at different concentration, essentially because of radiation damage issues. Hope these advices may help you. Regards, Marco --- Marco Mazzorana, PhD Structural Biology and Biocomputing Programme CNIO - Centro Nacional de Investigaciones Oncológicas C/ Melchor Fernández Almagro, 3, E-28029 Madrid (España) Phone: +34 91 2246900 ext. 3033 Fax: +34 91 2246976 www.cnio.es <http://www.cnio.es> --- Marjolein Thunnissen ha scritto: If you go to this web-page: http://www.embl-hamburg.de/ExternalInfo/Research/Sax/user_info.html there is ashort summary of the requirements for a SAXS experiment. Shortly the sample needs to be monodisperse (>90%) and you will need a range of concentrations roughly from 0.5/1.0mg/ml to 10mg/ml Marjolein Thunnissen Phone +46-(0)46-22 24584 Associate Professor Fax +46-(0)46-22 24692 Dept of Molecular Biophysics, Lund Universityhttp://www.mbfys.lu.se PO-Box 124 S-221 00 Lund, Sweden Scientific coordinator I911 (Max-lab): MAD and fixed-wavelength stations for macromolecular crystallography From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Li Sheng [biol...@gmail.com] Sent: Thursday, April 23, 2009 1:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein purity in SAXS Dear all, Is there anyone with experience in small angle x-ray scattering? Could anyone please tell me the requirements for protein sample purity and concentration? Thanx in advance. Sincerely, Li __ Email:s...@sibs.ac.cn Institute of Biochemistry and Cell Biology Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road, Shanghai 200031, China Tel: +86-21-5492-1217 __ **NOTA DE CONFIDENCIALIDAD** Este correo electrónico, y en su caso los ficheros adjuntos, pueden contener información protegida para el uso exclusivo de su destinatario. Se prohíbe la distribución, reproducción o cualquier otro tipo de transmisión por parte de otra persona que no sea el destinatario. Si usted recibe por error este correo, se ruega comunicarlo al remitente y borrar el mensaje recibido. **CONFIDENTIALITY NOTICE** This email communication and any attachments may contain confidential and privileged information for the sole use of the designated recipient named above. Distribution, reproduction or any other use of this transmission by any party other than the intended recipient is prohibited. If you are not the intended recipient please contact the sender and delete all copies.
Re: [ccp4bb] protein purity in SAXS
Dear Li, the samples should be devoid of aggregates, at least for the low concentration samples. Some aggregation can be tolerated at the higher concentrations. You should in principle measure samples with different concentrations, such as 1, 2, 5,10 mg/mL. The high concentration measurements will allow you to get a better signal/noise ratio at the higher s values. Cheers Isabelle **Dr. Isabelle Billas, PhD. Department of Structural Biology and Genomics Institute of Genetics and of Molecular and Cellular Biology IGBMC/ CNRS(UMR 7104) / INSERM (U964)/UDS/Collège de France CEBGS 1, rue Laurent Fries BP 10142 67404 ILLKIRCH CEDEX FRANCE Li Sheng a écrit : Dear all, Is there anyone with experience in small angle x-ray scattering? Could anyone please tell me the requirements for protein sample purity and concentration? Thanx in advance. Sincerely, Li __ Email:s...@sibs.ac.cn Institute of Biochemistry and Cell Biology Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road, Shanghai 200031, China Tel: +86-21-5492-1217 __
Re: [ccp4bb] protein purity in SAXS
If you go to this web-page: http://www.embl-hamburg.de/ExternalInfo/Research/Sax/user_info.html there is ashort summary of the requirements for a SAXS experiment. Shortly the sample needs to be monodisperse (>90%) and you will need a range of concentrations roughly from 0.5/1.0mg/ml to 10mg/ml Marjolein Thunnissen Phone +46-(0)46-22 24584 Associate Professor Fax +46-(0)46-22 24692 Dept of Molecular Biophysics, Lund Universityhttp://www.mbfys.lu.se PO-Box 124 S-221 00 Lund, Sweden Scientific coordinator I911 (Max-lab): MAD and fixed-wavelength stations for macromolecular crystallography From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Li Sheng [biol...@gmail.com] Sent: Thursday, April 23, 2009 1:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein purity in SAXS Dear all, Is there anyone with experience in small angle x-ray scattering? Could anyone please tell me the requirements for protein sample purity and concentration? Thanx in advance. Sincerely, Li __ Email:s...@sibs.ac.cn Institute of Biochemistry and Cell Biology Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road, Shanghai 200031, China Tel: +86-21-5492-1217 __
[ccp4bb] protein purity in SAXS
Dear all, Is there anyone with experience in small angle x-ray scattering? Could anyone please tell me the requirements for protein sample purity and concentration? Thanx in advance. Sincerely, Li __ Email:s...@sibs.ac.cn Institute of Biochemistry and Cell Biology Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road, Shanghai 200031, China Tel: +86-21-5492-1217 __
