Re: [ccp4bb] Off-topic: Native gel electrophoresis of basic proteins

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Summary: Dry shippers
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-08-27

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Subject: Re: Off-topic: Native gel electrophoresis of basic proteins
From: Vangelis Christodoulou e {- dot -} christodoulou {- at -} NKI {- dot -} NL
Date: 2008-08-27

Subject: Re: Off-topic: Native gel electrophoresis of basic proteins
From: Steven Darnell sdarnell {- at -} BIOCHEM {- dot -} WISC {- dot -} EDU
Date: 2008-08-27

Ming,

On that note, here is a Blue Native PAGE protocol that reportedly
improves the separation of basic proteins. A friend and I have used it
for native protein separation, but not necessarily for basic proteins.
Word of warning: don't use histidine salts or Tris-HCl in the cathode
buffer. The Figure 1 caption mentions this buffer shouldn't have
chloride ions in it.

Regards,
Steve Darnell

> Electrophoresis. 2006 Oct;27(20):3949-51.
>
> Discontinuous native protein gel electrophoresis.
>
> Niepmann M, Zheng J.
>
> Institute of Biochemistry, Justus-Liebig-University Giessen, Giessen,
> Germany.
> michael.niepmann@biochemie.med.uni-giessen.de
>
> Analysis of the oligomeric state of a native protein usually requires
> analytical
> ultracentrifugation or repeated gel filtration to calculate the
> protein's size.
> We have developed a discontinuous native protein gel electrophoresis
> system that
> allows the separation of even basic proteins according to their size,
> oligomeric
> state, and shape. This gel system combines the addition of negative
> charges to
> the proteins by Serva Blue G with a discontinuous buffer system and
> gradient
> gels. As in SDS-PAGE, chloride constitutes the high mobility anion in
> the gel and
> anode buffer. However, for sample focusing this system employs
> histidine instead
> of glycine as the slow dipolar ion following from the cathode buffer
> to improve
> migration of basic proteins. In addition, proteins run into gel pores
> corresponding to their size and shape in the gradient gel. Using this
> gel system,
> we show that the polypyrimidine tract-binding protein (PTB) is a monomer.
>
>
> PMID: 16991206 [PubMed - indexed for MEDLINE]

--
Steven Darnell
Department of Biochemistry
University of Wisconsin-Madison
Madison, WI USA

artem@XTALS.ORG said the following on 8/26/08 12:20 PM:
> You could try Coomassie Blue Native gel. It's a very neat technique and it
> worked for me on a couple of occasions. In one unfortunate case, it
> resulted in dissociation of a heterotetrameric complex, though.
>
> Artem
>
>
>> Dear CCP4 community,
>>
>> Sorry for the off-topic subject, but I would really appreciate some
>> suggestions and/or protocols relating to native gel electrophoresis of
>> basic proteins. I have used a general acidic PAGE protocol for my protein,
>> which has a PI of 9.5. Briefly, the protein was loaded onto a native gel
>> (I have tried both the pre-made Biorad gels (7.5% and a gradient gel:
>> 4-15%) and freshly prepared Tris native gels adjusted to pH 6.8) and run
>> in a 1X acetic acid/b-alanine pH 4.5 running buffer. The electrodes were
>> reversed and the gel run on ice for ~ 2hrs at 100V. In all cases, the
>> native protein was unable to enter the gel. Some protein samples incubated
>> with heavy atoms were able to enter the gel (possibly indicating binding)
>> but these samples too had problems entering the gel as the bands were at
>> or just a little bit below the edge of the well. Any suggestions and
>> comments would be most welcome!
>>
>> Thank you so much in advance for your help,
>> Sincerely,
>> Ming Lye
>>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Summary: Dry shippers
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-08-27

Next message:
Subject: Re: Off-topic: Native gel electrophoresis of basic proteins
From: Vangelis Christodoulou e {- dot -} christodoulou {- at -} NKI {- dot -} NL
Date: 2008-08-27