Re: [ccp4bb] Native Gel Charge States Vs. Conformations Vs. Oligomeric States

- Protein crystallography

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

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From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
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Subject: Liquid liquid phase separation
From: Debajyoti Dutta debajyoti_dutta47 {- at -} REDIFFMAIL {- dot -} COM
Date: 2009-01-17

Subject: Re: Native Gel Charge States Vs. Conformations Vs. Oligomeric States
From: Zhijie LI zhijie {- dot -} li {- at -} UTORONTO {- dot -} CA
Date: 2009-01-16

Hello Jacob,

The problem for native gel is that it is much more sensitive to a
single charge difference than size differences. Also, the gel pattern
may change greatly if you use a different buffer system. I had a case
2 years ago that my protein ran at 5 positions on a Laemmli(pH 8.8)
native gel, but only one position on a pH 7 gel. This protein only has
one clean peak corresponding to the monomer size on SEC when loaded at
1mg/mL, pH 6.8. So I believe what I was seeing on the pH 8.8 gel was
either some small conformational inhomogeneities(causing charged side
chains to be more exposed or buried) or some kind of aggregation
caused by the rather basic pH in the gel system - or even deamination
or decarboxylations... maybe.

As an inexpensive alternative to normal native gels, there is another
technique you might want to try: the blue native gel. In the blue
native gels, the protein's charge will mainly come from bound
Commassie Blue G250 molecues, so the migrating pattern should only
reflect the size of the protein particle. But the downside is that you
may have to spend some time to figure out your own protocol for your
particular protein, since some protein may precipitate or aggregate
badly with Commassie Blue G250 (and even worse if you use R250, which
has ethyl instead of methyl groups).

If you have access to a DLS machine, it might be a much better judge
for the disagreement between your SEC and AUC results.

Zhijie

Quoting Jacob Keller :

> Dear Crystallographers,
>
> I am having trouble interpreting the attached native gel (which is very
> repeatable, under various conditions). Is it tenable that various
> charge states or conformations could account for this behavior, or must
> it be various oligomeric states? AUC results seem to show only
> monomers, whereas SEC shows a series of peaks. Mass-spec shows no
> post-translational mods. Anyone have any similar experiences, or
> references?
>
> Thanks,
>
> Jacob
>
> *******************************************
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> Dallos Laboratory
> F. Searle 1-240
> 2240 Campus Drive
> Evanston IL 60208
> lab: 847.491.2438
> cel: 773.608.9185
> email: j-keller2@northwestern.edu
> *******************************************

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

Next message:
Subject: Liquid liquid phase separation
From: Debajyoti Dutta debajyoti_dutta47 {- at -} REDIFFMAIL {- dot -} COM
Date: 2009-01-17