Hi Rich,
I'm not sure but I imagine it would also apply to single span proteins. I have never encountered a helical membrane protein that could be boiled w/o aggregating, even very stable ones. Of course, a more elaborate sample preparation involving boiling, sonication and urea as mentioned by Ruud might work for some proteins. Bert ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Richard Berry <richard.be...@monash.edu> Sent: 02 March 2017 05:50 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Dimer in SDS-PAGE Dear Bert You made the comment a few weeks ago not to boil helical membrane proteins for SDS-PAGE. Could i please ask, does this also apply to type I membrane proteins that only have a single a-helix, or is it just membrane proteins that are predominantly helical? Thanks Rich On 22 February 2017 at 19:18, Bert Van-Den-Berg <bert.van-den-b...@newcastle.ac.uk<mailto:bert.van-den-b...@newcastle.ac.uk>> wrote: like others I'm not clear why you care where your protein runs on SDS-PAGE. I think the band you're seeing is in fact the tetramer, suggesting your protein (like KcsA) is very stable. Helical membrane proteins often migrate faster than expected (by their Mw) on SDS-PAGE. Also, never boil helical membrane protein samples, they will aggregate. bert ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of amit gaur <cdriamitg...@gmail.com<mailto:cdriamitg...@gmail.com>> Sent: 21 February 2017 22:22 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Dimer in SDS-PAGE Hi all, I am trying to purify a potassium ion channel from insect cell using baculovirus expression system. I am not seeing monomer of this protein in SDS instead a dimer appears.So,I increased DTT in SDS buffer but no change and dimer was intact. In size exclusion this protein appeared as a tetramer which is common oligomerizaton of potassium channel family with GYG motif. Can any body suggest what should I do in this case? Thanks and regards, -- Dr. Amit Gaur Post Doctoral Researcher PI: Dr. Ji-Fang Zhang Thomas Jefferson University 1020, Locust Street, Suite 418 Philadelphia, PA 19107 -- -- Dr Richard Berry NHMRC Career Development Fellow Department of Biochemistry and Molecular Biology Monash University Ground Floor, Building 76, ClaytonCampus Blackburn Road Clayton VIC 3800 Australia T: +61 3 9902 9239 E: richard.be...@monash.edu<mailto:name.surn...@monash.edu>
