[Protein-analysis] heterooligomer or homooligomer

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[Protein-analysis] heterooligomer or homooligomer

SL-5
Hi,

 From gel filtration + western, my protein looks much bigger (~ 200 kD)
than its monomer size (~60 kD). I am just wondering if there is a method
I can detect if it's a protein complex (with other proteins), or just a
oligomer of itself.

Any suggestions are welcome.

Thanks.

LS
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[Protein-analysis] Re: heterooligomer or homooligomer

SL-5
I mean to know this before I am starting to purify this complex.

thanks.

ls

SL wrote:

> Hi,
>
>  From gel filtration + western, my protein looks much bigger (~ 200 kD)
> than its monomer size (~60 kD). I am just wondering if there is a method
> I can detect if it's a protein complex (with other proteins), or just a
> oligomer of itself.
>
> Any suggestions are welcome.
>
> Thanks.
>
> LS
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[Protein-analysis] Re: heterooligomer or homooligomer

SL-2
In reply to this post by SL-5
Yes.

Factions were collected from gel filtration, and then each fraction was
loaded onto SDS-PAGE for western. Western showed my protein in one
fraction and the size is about 60 kD. But from gel filtration, that
fraction should be about 200 kD.


subberry wrote:
> is there a SDS-PAGE before western?
>
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[Protein-analysis] Re: heterooligomer or homooligomer

Nick Theodorakis
In reply to this post by SL-5
On Thu, 17 Nov 2005 17:59:02 -0500, SL <[hidden email]> wrote:

>SL wrote:
>> Hi,
>>
>>  From gel filtration + western, my protein looks much bigger (~ 200 kD)
>> than its monomer size (~60 kD). I am just wondering if there is a method
>> I can detect if it's a protein complex (with other proteins), or just a
>> oligomer of itself.
>>

>I mean to know this before I am starting to purify this complex.
>

It's going to be difficult to answer that question definitively
without further purification. If it's abundant enough, and you have an
antibody, you could something like an immuniprecipation followed by
examination of the pellet by PAGE (either 1D or 2D), but it will take
a lot of work to rule out non-specific binding.

The answer will most likely become clearer during purification, but
since you are going to purify it anyway, why is it so important to
know before you start? Arthur Kornberg once said, "don't waste clean
thinking on dirty enzymes."

Nick

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Nick Theodorakis
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[Protein-analysis] Re: heterooligomer or homooligomer

Bjoern-7
In reply to this post by SL-5
Well you are right, sorry. What I meant was non-reducing. That means
one sample with and one withoue DTT or mercaptoethanol.

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[Protein-analysis] Re: heterooligomer or homooligomer

Dr Engelbert Buxbaum
In reply to this post by SL-5
[hidden email] wrote:


> "SDS under non-denaturing conditions" the most beautiful oxymorone I've
> ever heard.

Not really. If the sample is gently heated in the presence of SDS, but
absence of sulphhydryls, some (but not all) protein secondary and
tertiary structure as well as protein-proteins interactions are
maintained. Of course, even milder  methods are also available, DISK-
and native-blue electrophoresis are but two. Indeed, it is possible to
do a non-denaturing electrophoresis in the first and a denaturing in the
second dimension. This is a very useful method to study protein-protein
interactions.
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