Re: [ccp4bb] Separating Monomers and Dimers
Sorry if this is an insulting question, but did you store it in enough glycerol to prevent it from freezing? Proteins don't like freeze/thaw cycles, especially slow ones. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of jai mohan [0cab66323371-dmarc-requ...@jiscmail.ac.uk] Sent: Tuesday, June 27, 2017 7:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Separating Monomers and Dimers Dear all, I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 27kDa. After purification I ran a SDS page, the band at 27kDa confirms the monomer. The protein was stored at -20C, a week later again I ran a gel, this time I saw another new band between 50-60kDa, it confirms the protein solution contains both monomers and dimers. I would like to know, what is the best way to separate the monomers and dimers? One of my colleague advice me to go for sucrose gradient centrifugation and size exclusion chromatography. However, I seek all your valuable suggestions and advice. With best regards Dr. S.M.Jaimohan
Re: [ccp4bb] Separating Monomers and Dimers
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 <0cab66323371-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
Re: [ccp4bb] Separating Monomers and Dimers
Hello Jaimohan, Is it SDS-PAGE gel or Native PAGE gel ?. Theoretically SDS-PAGE is supposed to show monomers and Native PAGE oligomers On Tue, Jun 27, 2017 at 8:27 AM jai mohan < 0cab66323371-dmarc-requ...@jiscmail.ac.uk> wrote: > Dear all, > I am working on a Red Fluorescent Protein (His-Tag) molecular weight > around 27kDa. After purification I ran a SDS page, the band at 27kDa > confirms the monomer. The protein was stored at -20C, a week later again I > ran a gel, this time I saw another new band between 50-60kDa, it confirms > the protein solution contains both monomers and dimers. I would like to > know, what is the best way to separate the monomers and dimers? One of my > colleague advice me to go for sucrose gradient centrifugation and size > exclusion chromatography. > However, I seek all your valuable suggestions and advice. > > With best regards > Dr. S.M.Jaimohan >
Re: [ccp4bb] Separating Monomers and Dimers
Are you boiling your samples? Seems funny that under SDS PAGE there should be dimers, unless there is a disulfide link. If so, reducing agents (DTT, TCEP, BME) should take care of this. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Smith Liu Sent: Tuesday, June 27, 2017 8:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Separating Monomers and Dimers it was not stable for frozen storage. if necessary，using protein fresh without frozen 发自网易邮箱大师 在2017年06月27日 20:22，jai mohan<mailto:0cab66323371-dmarc-requ...@jiscmail.ac.uk> 写道： Dear all, I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 27kDa. After purification I ran a SDS page, the band at 27kDa confirms the monomer. The protein was stored at -20C, a week later again I ran a gel, this time I saw another new band between 50-60kDa, it confirms the protein solution contains both monomers and dimers. I would like to know, what is the best way to separate the monomers and dimers? One of my colleague advice me to go for sucrose gradient centrifugation and size exclusion chromatography. However, I seek all your valuable suggestions and advice. With best regards Dr. S.M.Jaimohan
Re: [ccp4bb] Separating Monomers and Dimers
it was not stable for frozen storage. if necessary，using protein fresh without frozen 发自网易邮箱大师 在2017年06月27日 20:22，jai mohan 写道： Dear all, I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 27kDa. After purification I ran a SDS page, the band at 27kDa confirms the monomer. The protein was stored at -20C, a week later again I ran a gel, this time I saw another new band between 50-60kDa, it confirms the protein solution contains both monomers and dimers. I would like to know, what is the best way to separate the monomers and dimers? One of my colleague advice me to go for sucrose gradient centrifugation and size exclusion chromatography. However, I seek all your valuable suggestions and advice. With best regards Dr. S.M.Jaimohan
