Re: [ccp4bb] A question on protein microheterogenity for crystalization
We had this happen with RPA14/32 heterodimer, we kept the two peaks separate, and then grew crystals from each but in different space groups and diffraction resolution. See Habel, J. E., Ohren, J. F. and Borgstahl, G. E. O. "Dynamic light scattering analysis of full-length, human RPA14/32 dimer: purification crystallization and self-association" *Acta Cryst.*D57, 254-259 (2001). On Sun, Dec 15, 2013 at 2:33 PM, Phoebe A. Rice wrote: > It might be that the protein is innocent and the FPLC is guilty. If > FPLC pump needs maintenance and is stuttering a bit when running at high > pressure, the salt gradient won't be as smooth as you'd like. If you have > an ionic strength detector, that trace will tell you. Otherwise, see if > other people's proteins are also looking a bit iffy under similar > circumstances. > Phoebe > -- > *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of > Katherine Sippel [katherine.sip...@gmail.com] > *Sent:* Sunday, December 15, 2013 11:53 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* Re: [ccp4bb] A question on protein microheterogenity for > crystalization > > Hi Acoot, > > There are a lot of great suggestions here already. I've also run into this > phenomenon in couple of cases. The first was a binding protein in mixed > bound and unbound forms. The second was a case of heterogeneous > post-translational modification. In both cases I could only get crystals > from purified peaks and not pooled protein. If protein is precious I'd > second Mark's suggestion to try screening pooled protein while you scale up > your prep. > > Cheers, > Katherine > > > On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett wrote: > >> Dear All, >> >> When I purified my protein by ion exchange chromatography for >> crystallization, there were several peaks containing the target protein as >> analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel >> filtration coupled MALLS. >> >> For crystallization purpose, can I merge the corresponding ion exchange >> chromatography peaks together? Otherwise the protein yield will be too low. >> And how to explain the heterogeneity by ion exchange chromatography in this >> situation? >> >> I am looking forward to getting a reply from you. >> >> Acoot >> > > > > -- > "Nil illegitimo carborundum"* - *Didactylos >
Re: [ccp4bb] A question on protein microheterogenity for crystalization
It might be that the protein is innocent and the FPLC is guilty. If FPLC pump needs maintenance and is stuttering a bit when running at high pressure, the salt gradient won't be as smooth as you'd like. If you have an ionic strength detector, that trace will tell you. Otherwise, see if other people's proteins are also looking a bit iffy under similar circumstances. Phoebe From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Katherine Sippel [katherine.sip...@gmail.com] Sent: Sunday, December 15, 2013 11:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization Hi Acoot, There are a lot of great suggestions here already. I've also run into this phenomenon in couple of cases. The first was a binding protein in mixed bound and unbound forms. The second was a case of heterogeneous post-translational modification. In both cases I could only get crystals from purified peaks and not pooled protein. If protein is precious I'd second Mark's suggestion to try screening pooled protein while you scale up your prep. Cheers, Katherine On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett mailto:acootbr...@yahoo.com>> wrote: Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot -- "Nil illegitimo carborundum" - Didactylos
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot, There are a lot of great suggestions here already. I've also run into this phenomenon in couple of cases. The first was a binding protein in mixed bound and unbound forms. The second was a case of heterogeneous post-translational modification. In both cases I could only get crystals from purified peaks and not pooled protein. If protein is precious I'd second Mark's suggestion to try screening pooled protein while you scale up your prep. Cheers, Katherine On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett wrote: > Dear All, > > When I purified my protein by ion exchange chromatography for > crystallization, there were several peaks containing the target protein as > analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel > filtration coupled MALLS. > > For crystallization purpose, can I merge the corresponding ion exchange > chromatography peaks together? Otherwise the protein yield will be too low. > And how to explain the heterogeneity by ion exchange chromatography in this > situation? > > I am looking forward to getting a reply from you. > > Acoot > -- "Nil illegitimo carborundum"* - *Didactylos
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot: Can your protein form some kind of dynamic self oligomers? When you test by using the gel-filtration column, if the peak is symmetry? Sometime if the target protein can have week self interaction, you can observe a tailed peak. If the protein can have a strong self interaction, maybe you can observe isolated peaks, each peak is corresponding to different amount of the oligomersation state of the protein. You also can test your protein by using native or IEF gels. Dee Date: Sat, 14 Dec 2013 19:01:09 +0100 From: mjvanra...@cnb.csic.es Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization To: CCP4BB@JISCMAIL.AC.UK Dear Brett, We have seen this behaviour several times for different adenovirus fibre head proteins and don't really have an explanation for it. We have always set the peaks up separately when we had enough protein. For this purification, as you don't have enough protein to pool them separately, I would pool them together and do a first crystallisation screen. But I would also immediately start a larger scale purification so you in the next crystallisation trial you can set the peaks up separately. If you are lucky, by the time you have done the second prep, from the first screen you may have some conditions to optimise. If not, the first screen should at least give some ideas about which precipitants, pHs and perhaps additives are most suitable.Mark On 14 Dec 2013, at 13:16, Acoot Brett wrote:Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Dear Brett, We have seen this behaviour several times for different adenovirus fibre head proteins and don't really have an explanation for it. We have always set the peaks up separately when we had enough protein. For this purification, as you don't have enough protein to pool them separately, I would pool them together and do a first crystallisation screen. But I would also immediately start a larger scale purification so you in the next crystallisation trial you can set the peaks up separately. If you are lucky, by the time you have done the second prep, from the first screen you may have some conditions to optimise. If not, the first screen should at least give some ideas about which precipitants, pHs and perhaps additives are most suitable. Mark On 14 Dec 2013, at 13:16, Acoot Brett wrote: > Dear All, > > When I purified my protein by ion exchange chromatography for > crystallization, there were several peaks containing the target protein as > analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel > filtration coupled MALLS. > > For crystallization purpose, can I merge the corresponding ion exchange > chromatography peaks together? Otherwise the protein yield will be too low. > And how to explain the heterogeneity by ion exchange chromatography in this > situation? > > I am looking forward to getting a reply from you. > > Acoot
Re: [ccp4bb] A question on protein microheterogenity for crystalization
Hi Acoot, since they behave differently on IEX, they are different - you would introduce heterogeneity into your crystallization setup, which usually is not a good idea for crystallization in general. Are you using Benzonucleases by any chance in your preparation and if not, that may explain the different populations of your protein. Your protein may bind some short DNA chunks carried over during your lysis process leading to differently charged species on the IEX column. On an SDS gel these complexes are separated and your protein runs at an identical molecular weight. An alternative explanation would be multiple folded states that may be at equilibrium and under certain conditions e.g. pH, salt concentration may be pushed in one predominant species. Also is several 2 or 10 peaks ? If you have e.g. an N-terminal His6-tag you might end up with truncation products and say your protein is rather large e.g. 80 kDa you might not be able to see the molecular weight difference either by SEC or SDS-PAGE if you are running a low percentage gel. A protein of 72 kDa might look like 80 kDa but you most likely will have charge differences distinguishable in IEX. If you have a His6-tag, run a Western on it to identify single or multiple bands. Good luck, Jürgen On Dec 14, 2013, at 7:16 AM, Acoot Brett mailto:acootbr...@yahoo.com>> wrote: Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot .. 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] A question on protein microheterogenity for crystalization
Dear All, When I purified my protein by ion exchange chromatography for crystallization, there were several peaks containing the target protein as analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel filtration coupled MALLS. For crystallization purpose, can I merge the corresponding ion exchange chromatography peaks together? Otherwise the protein yield will be too low. And how to explain the heterogeneity by ion exchange chromatography in this situation? I am looking forward to getting a reply from you. Acoot
