Re: [ccp4bb] Dimer in SDS-PAGE

[Bert Van-Den-Berg]

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>

Re: [ccp4bb] Dimer in SDS-PAGE

[Ruud Hovius]

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 
<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>


--

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

[Richard Berry]

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 <CCP4BB@JISCMAIL.AC.UK> on behalf of amit
> gaur <cdriamitg...@gmail.com>
> *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 <name.surn...@monash.edu>

Re: [ccp4bb] Dimer in SDS-PAGE

[Bert Van-Den-Berg]

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> on behalf of amit gaur 
<cdriamitg...@gmail.com>
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

[Dr. Isabel De Moraes]

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 
> 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





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Re: [ccp4bb] Dimer in SDS-PAGE

[Mark Brooks]

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

[Keller, Jacob]

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

[amit gaur]

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*