Dear Jaimohan, With high amount of purified protein (like obtained in recombinant expression) they are likely to form higher oligomers if they have intrinsic property of association. With time the content of oligomers do increase. In SDS-PAGE even, you will see a faint band of oligomer. This is due to higher mass of protein from which some still could form oligomer even after boiling and presence of SDS. I am sure your protein has higher content of helical fraction. My suggestion to you would be to perform HPLC or FPLC every time before assay or exp. It would reliably separate the monomer and higher oligomers.
Best regards, Debasish Kumar Ghosh CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html ----- Original Message ----- From: jai mohan <00000cab66323371-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 27 Jun 2017 17:52:34 +0530 (IST) Subject: [ccp4bb] Separating Monomers and Dimers Dear all,I am working on a Red FluorescentProtein (His-Tag) molecular weight around 27kDa. After purification I ran a SDSpage, the band at 27kDa confirms the monomer. The protein was stored at -20C, aweek later again I ran a gel, this time I saw another new band between 50-60kDa, it confirmsthe protein solution contains both monomers and dimers. I would like to know, whatis the best way to separate the monomers and dimers? One of my colleague adviceme to go for sucrose gradient centrifugation and size exclusion chromatography.However, I seek all your valuable suggestions and advice. With best regardsDr. S.M.Jaimohan
