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At 11:56 AM 4/12/2011, Lake Paul wrote:<br>
<blockquote type=cite class=cite cite="">Folks,<br>
I have a sample, a membrane protein with 0.01% DDM. I collected data at
45000 rpm, 4 min/scan. The sample is very heterogeneous but I am getting
a decent peak from the absorbance data (280 nm) at 7.4 S, but the
interference data gives me a peak at 6.5 S. I am trying to calculate the
amount of detergent bound so I need to align the interference data with
absorbance data. </blockquote><br>
<blockquote type=cite class=cite cite="">This is the first time in which
I have gotten the peaks from the interference data not lining up with
absorbance data. </blockquote><br>
First time in general, or first time for a membrane protein?<br><br>
When you say "heterogenous" what exactly do you mean by
that?<br><br>
I had never collected data by AUC, but I would guess that size
exclusion chromatography with absorbance, refractometry and light
scattering detection will shed light on the state of this sample.
>From triple detection in SEC-LS/UV/RI I had seen cases where
refraction will not align with absorbance for mombrane protein complexes.
<br><br>
Ewa<br><br>
<blockquote type=cite class=cite cite="">Is this due to the time it takes
to make an absorbance scan relative to the interference scans? And/or I
just spun the sample too hard? Or is the DDM affecting the sedimentation?
See the attached powerpoint. <br>
Yes, the slit assembly is functioning properly J <br>
Any suggestions will be helpful! <br>
Best,<br>
Lake<br>
<br>
PS I collected data during the same run with the same sample except it
had GFP attached. The interference peak shifted accordingly. The DDM peak
lined up well also. <br>
<br>
<b><i>Lake N. Paul, PhD<br>
</i></b>Biophysics Research Specialist<br>
Discovery Park - Bindley Biosciences Center Purdue University<br>
1203 West State Street<br>
West Lafayette, Indiana, 47905<br>
765.494.4960<br>
<b><u>BioAnalytical Lab Website:<br>
</u></b>
<a href="http://www.purdue.edu/discoverypark/bioanalytical/" eudora="autourl">
http://www.purdue.edu/discoverypark/bioanalytical/</a><br>
<br><br>
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