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> 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> 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 >> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > 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 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/
