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<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>Hej Paul, </SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>looking at your data, there are several
observations:</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>- the meniscii from Abs and Int are not equal (ca. 5.97
cm for Int and ca. 5.93 cm for Abs). You might want to compare the radial
calibrations from Abs and Int by scanning the CB. If you can't get them to
overlap yourself, ask your field technician to do it. Distorted radial
calibrations will give distorted data, will give distorted fits,
etc.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>- from the Int data, it looks as if you have at least
three fractions present, two for the high s-region, one for the low s. The high
s-range looks similar for Int and Abs (and your Abs-c(s) does indeed show two
signals), so your Int c(s)-result seems too detailled for the amount of
data/information in your sample. You might want to try a "simpler" approach like
dc/dt or vHW.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>- it looks as if the high s-region in your Int data is
completely separarated from your low s-region. You could therefore try to
determine the concentration of material in that region simply by taking the
signal amplitude of the first plateau and extrapolate that to wexp2t=0. The same
procedure for Abs would give you a handle on the signal of detergent
co-sedimenting. From my experience however, it's a good idea to perform
independent measurements of the single compounds for such kind of
calculations.</SPAN></FONT></DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011></SPAN></FONT> </DIV>
<DIV dir=ltr align=left><FONT face=Verdana color=#0000ff size=2><SPAN
class=582043810-13042011>Best, Holger</SPAN></FONT></DIV><BR>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left>
<HR tabIndex=-1>
<FONT face=Tahoma size=2><B>From:</B> rasmb-bounces@rasmb.bbri.org
[mailto:rasmb-bounces@rasmb.bbri.org] <B>On Behalf Of </B>Lake
Paul<BR><B>Sent:</B> Tuesday, April 12, 2011 5:57 PM<BR><B>To:</B>
rasmb@rasmb-email.bbri.org<BR><B>Subject:</B> [RASMB] Speed of Interference
Optics vs. Absorbance Optics<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV class=WordSection1>
<P class=MsoNormal>Folks,<o:p></o:p></P>
<P class=MsoNormal>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. This is the first time in which I have gotten the peaks from the
interference data not lining up with absorbance data. 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. <o:p></o:p></P>
<P class=MsoNormal>Yes, the slit assembly is functioning properly <SPAN
style="FONT-FAMILY: Wingdings">J</SPAN> <o:p></o:p></P>
<P class=MsoNormal>Any suggestions will be helpful! <o:p></o:p></P>
<P class=MsoNormal>Best,<o:p></o:p></P>
<P class=MsoNormal>Lake<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>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. <o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><B><I><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Lake N. Paul,
PhD<o:p></o:p></SPAN></I></B></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Biophysics Research
Specialist<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Discovery Park - Bindley
Biosciences Center Purdue University<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">1203
West State Street<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">West
Lafayette, Indiana, 47905<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">765.494.4960<o:p></o:p></SPAN></P>
<P class=MsoNormal><B><U><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">BioAnalytical Lab
Website:<o:p></o:p></SPAN></U></B></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">http://www.purdue.edu/discoverypark/bioanalytical/<o:p></o:p></SPAN></P>
<P class=MsoNormal><o:p> </o:p></P></DIV></BODY></HTML>