Re: [ccp4bb] Dimer in SDS-PAGE
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 on behalf of Richard Berry 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 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 mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of amit gaur 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>
Re: [ccp4bb] Dimer in SDS-PAGE
Dear all, this all depends on the protein: some 7TM proteins are nicely resolved as 1 band of ~the expected size upon 5 min sonication - 5 min 95 °c - 5 min sonication in sample buffers containing 2 M urea Others still gave mono- & dimers The treatment above also wored for a pentameric 4TM protein Detection by commassie or silver stain for bulk & autoradiography for 35S-Met pulse labelling. You have to try your luck. Best greetings, Ruud On 2/3/17 06:50, Richard Berry wrote: 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 <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 mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of amit gaur 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> -- Ruud Hovius EPFL SB ISIC LIP BCH 4209 CH-1015 Lausanne +41-21-693-9442 http://lip.epfl.ch
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> 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 on behalf of amit > gaur > *Sent:* 21 February 2017 22:22 > *To:* 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
Re: [ccp4bb] Dimer in SDS-PAGE
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 on behalf of amit gaur Sent: 21 February 2017 22:22 To: 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
Re: [ccp4bb] Dimer in SDS-PAGE
Dear Amit, You are working with a membrane protein and the use of SDS (harsh detergent) often makes these proteins to oligomerise. Boiling your sample is not advisable, it might make it worse. I’m not sure why you would like to “see” a monomer in the gel but if you really would like to know what is the oligomeric state of you protein in solution, I would advise you to do a SEC-MALS (size exclusion chromatography - multi angle light scattering) run of your sample. Best, Isabel - Dr Isabel Moraes Membrane Protein Laboratory Group Leader Diamond Light Source Ltd, Harwell Science and Innovation Campus, Oxfordshire, OX11 ODE, UK - On 21 Feb 2017, at 22:22, amit gaur mailto:cdriamitg...@gmail.com>> wrote: 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 -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] Dimer in SDS-PAGE
Dear Amit, Maybe try adding an equal volume of 8M urea to your sample before adding the SDS-PAGE sample buffer. Then I'd test boiling and not boiling that sample prior to loading on the gel. Good luck, Mark On 21 February 2017 at 17:22, amit gaur wrote: > 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 ResearcherPI: Dr. Ji-Fang ZhangThomas Jefferson University* > *1020, Locust Street, Suite 418* > *Philadelphia, PA 19107* > > >
Re: [ccp4bb] Dimer in SDS-PAGE
Why do you need to “do” anything? Is there some reason you would like to see a monomer on your gels? It is common for membrane proteins to run as non-covalent oligomers in SDS-PAGE, and sometimes boiling makes it even worse. I think KCSA runs as a tetramer on gels, and several proteins I have worked with have never run exclusively (or at all!) as monomers, but often ran as ladders depending on conditions. On the topic of unusual SDS-PAGE phenomena, it might be of general interest to point out that GFP and proteins tagged therewith remain fluorescent in SDS-PAGE gels unless they are boiled. And…RFP (mRuby2 in my case) seems to remain fluorescent even after boiling, although it shifts its apparent MW. And, well, two more: phosphorylation shifts apparent MW way more than the phosphate group’s mass, and calmodulin and other calcium-binding proteins shift a lot +/- calcium. There are of course explanations ex post facto, but I guess the general idea is that SDS-PAGE does not always work as the textbooks say. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of amit gaur Sent: Tuesday, February 21, 2017 5:23 PM To: 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
[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 ResearcherPI: Dr. Ji-Fang ZhangThomas Jefferson University* *1020, Locust Street, Suite 418* *Philadelphia, PA 19107*