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
<[email protected]
<mailto:[email protected]>> 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 <[email protected]
<mailto:[email protected]>> on behalf of amit gaur
<[email protected] <mailto:[email protected]>>
*Sent:* 21 February 2017 22:22
*To:* [email protected] <mailto:[email protected]>
*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: [email protected] <mailto:[email protected]>
--
Ruud Hovius
EPFL SB ISIC LIP
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CH-1015 Lausanne
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http://lip.epfl.ch
