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Karen's comment: <br><br>
<br>
<blockquote type=cite class=cite cite="">The other issue is that membrane
proteins cannot necessarily be thought of as binding a "micelle's
worth" of detergent molecules. (See Marc Le Maire's work). This is
especially true when their molecular weights get larger. Therefore,
monomers and multimers may bind different numbers of detergent molecules
and therefore the buoyant molecular weight of the protein-detergent
complex may be different for monomers+detergent as compared to
multimers+detergent. We always got around this by density matching the
detergent, but sometimes that's not possible for the
system.</blockquote><br>
I would suggest running the size exclusion with light scattering,
refractometry and absorbance to dissect the contributions from protein
(polypeptide) and detergent for various oligomeric forms. <br><br>
1. Folta-Stogniew,E. 2006. Oligomeric states of
proteins determined by size-exclusion chromatography coupled with light
scattering, absorbance, and refractive index detectors. <i>Methods Mol.
Biol.</i> 328:97-112.<br>
2. Hayashi,Y., H.Matsui, and T.Takagi. 1989.
Membrane protein molecular weight determined by low-angle laser
light-scattering photometry coupled with high-performance gel
chromatography. <i>Methods in Enzymology</i> 172:514-528.<br>
3. Wei,Y., H.Li, and D.Fu. 2004. Oligomeric
State of the Escherichia coli Metal Transporter YiiP. <i>J. Biol.
Chem.</i> 279:39251-39259.<br>
4. Yernool,D., O.Boudker, E.Folta-Stogniew, and
E.Gouaux. 2003. Trimeric subunit stoichiometry of the glutamate
transporters from Bacillus caldotenax and Bacillus stearothermophilus.
<i>Biochemistry</i> 42:12981-12988.<br><br>
<br>
Ewa<br><br>
<br><br>
<blockquote type=cite class=cite cite="">
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<br><br>
On Nov 3, 2009, at 8:07 AM, Arthur Rowe wrote:<br><br>
<blockquote type=cite class=cite cite="">Hi John (and everyone)<br><br>
That sounds good to me. Of course you could have more than one protein
monomer contained in each micelle - but that should be immediately
obvious from the M value.<br><br>
Kind regards<br><br>
Arthur--<br>
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Arthur J Rowe<br>
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<dl>
<dd>From: <a href="mailto:John_Doran@vrtx.com">John_Doran@vrtx.com</a>
<dd>Date: Mon, 2 Nov 2009 16:46:47 -0500
<dd>To: rasmb@rasmb.bbri.org
<dd>Subject: [RASMB] Query concerning membrane protein sedimentation
equilibrium experiment<br><br>
</dl>
<dl>
<dd><font size=2>We have measured the amount of detergent bound to our
membrane protein and hence were able to calculate a Vbar for the protein
detergent complex. Is it sufficient to just substitute this value for
Vbar along with the density value of the detergent buffer solution into
the ideal species equation of heteroanalysis to get an accurate molecular
weight value of the protein-detergent complex, or is it more involved
than that? Any thoughts concerning this would be appreciated. Thank
you.</font> <br><br>
<dd><font size=2>John Doran</font>
<dd><font size=2>Vertex Pharmaceuticals</font>
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