It would be sensible to analyse the state of your purified protein further.
* (SEC in reasonable salt conc containing buffer – this is standard practice) * Analytical ion exchange of purified protein - are there different states? * SEC (in reasonable buffer) in line with MALS – is there a monomer-dimer equilibrium? * Analytical SEC (without further concentration) of the potential dimer side of the peak, repeat for monomer side – could give info on potential monomer-dimer equilibrium Cheers, Ross From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Javier Gonzalez Sent: 09 December 2020 14:52 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography [EXTERNAL SENDER] ________________________________ Hello, I agree with Roger, you should definitely try to increase the salt concentration to get rid of non specific binding impurities. And if that doesn't work, you can try purifying your protein under denaturing conditions by adding one refolding step in the column. Good luck, Javier On Wed, Dec 9, 2020 at 11:26 AM Roger Rowlett <rrowl...@colgate.edu<mailto:rrowl...@colgate.edu>> wrote: Salt concentrations less than 100 mM can lead to nonspecific adsorption to the gel exclusion media, potentially leading to band broadening, and delayed elution. Overloading gel exclusion columns (more than 2-4% Vt) can also lead to elution band artifacts. Check these issues first. Roger Rowlett Gordon & Dorothy Kline Professor, Emeritus Department of Chemistry Colgate University On Wed, Dec 9, 2020, 9:17 AM <MJ Xie> <sz20203020...@cau.edu.cn<mailto:sz20203020...@cau.edu.cn>> wrote: Dear All There is a 43kd protein purified via Ni-chelating affinity chromatography, anion exchange chromatography and gel filtration chromatography in sequence. However the chromatogram obtained showed an extremely asymmetric peak shape. The aggregation forms of proteins are mainly in the range of monomers and dimers(Hepes and low concentration of salt were used as buffers for gel filtration chromatography). 5% glycerinum and 1mM Benzamidine hydrochloride had been added in order to maintain the stability of the protein and prevent it from degrading. But well, all the efforts seem to be useless. We wonder if there are any effective measures can be taken to radically solve this problem. We would be much indebted for the suggestions you offer. ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> -- Dr. Javier M. González Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET) Universidad Nacional de Santiago del Estero (UNSE) RN9, Km 1125. Villa El Zanjón. (G4206XCP) Santiago del Estero. Argentina Tel: +54-(0385)-4238352 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> This message (and any associated files) is intended only for the use of the individual or entity to which it is addressed and may contain information that is confidential, subject to copyright or constitutes a trade secret. If you are not the intended recipient you are hereby notified that any dissemination, copying or distribution of this message, or files associated with this message, is strictly prohibited. If you have received this message in error, please notify us immediately by replying to the message and deleting it from your computer. Messages sent to and from us may be monitored. Internet communications cannot be guaranteed to be secure or error-free as information could be intercepted, corrupted, lost, destroyed, arrive late or incomplete, or contain viruses. Therefore, we do not accept responsibility for any errors or omissions that are present in this message, or any attachment, that have arisen as a result of e-mail transmission. If verification is required, please request a hard-copy version. Any views or opinions presented are solely those of the author and do not necessarily represent those of the company. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
