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<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>Hi Peter,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>in addition to the comments by John, some more from my
experience, mainly from SE:</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- purity is absolutely essential, esp. when working with
synthetic peptides. Ideally, measure the integrity of the peptide
with MALDI-TOF before and after the experiment.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- if at all possible, measure the vbar, the true value of
your peptide will likely deviate from the one Sednterp gives you. If you're only
after the stoichiometry of the complex, it might be sufficient to stick with
sigma (the lower limit of which you can define from the
data) and ratios thereof.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- you can check the reversibility of your reaction
from plots of lnC/r2 at different starting
concentrations. </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- the stoichiometry of the complex is more a problem of
accuracy, so you need the best data you can get; scan many replicates, use long
solution columns (200 microL and more), clean your lamp before the experiment,
use thoroughly dialysed buffer (2800 g/mole can be tough, though). The decision
wether your smallest sigma is really a monomer or a dimer is easier to answer
than wether you have a hexa- or a heptamer.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- try to measure the baseline absorbance. If you do have a
hexamer, you should be able to clear the meniscus with long solution columns at
the highest speeds. You can get the baseline for all wavelengths used from this
condition. </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- if you can clear the meniscus, interference has clear
advantages over absorbance (better S/N, mostly) and will help you to
define the upper limit of the association. Be careful to measure
a good water blank.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- use a buffer of sufficient ionic strength. Some peptides
carry surprisingly high charge densities.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>- if the peptide unfolds at low concentrations, degradation
can be a real problem. If so, </FONT></SPAN><SPAN class=473203808-12122011><FONT
face=Verdana color=#0000ff size=2>excluding as far as possible all sources
of microbial contamination can help, including rinsing your cells with 70%
ethanol.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=473203808-12122011><FONT face=Verdana
color=#0000ff size=2>Best, Holger</FONT></SPAN></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>Peter Edward Prevelige
Jr<BR><B>Sent:</B> Friday, December 09, 2011 11:57 PM<BR><B>To:</B>
rasmb@rasmb.bbri.org<BR><B>Subject:</B> [RASMB] Help with Small Self-Associating
Peptide<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV class=WordSection1>
<P class=MsoNormal>Hi All –<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Can you give me some advice on the best way to analyze a
small (~2800 Da) peptide that we think is self associating. The CD shows
increasing helicity with concentration which continues to increase even at 10
mg/ml meaning we will likely have to record data at a less than ideal wavelength
(240 nm) Our interest is really in determining the stoichiometry of the species
formed at high concentration (we suspect a hexamer), we care less about
Kd. Looking through the archives I’ve seen conflicting suggestions, (ie. sed
velocity vs sed equilibrium). I was thinking about mimicking the approach
in the Braswell/Holtzer paper but the peptide is somewhat valuable and limited
so I wanted to ascertain whether or not this was the optimal way to proceed.
<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Thanks!<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal>Peter<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Peter
E. Prevelige Jr.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Professor<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Dept.
of Microbiology, BBRB 416/6<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Univ.
of Alabama @ Birmingham<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">845
19th St. South<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Birmingham AL.
35294-2170<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">Phone
205 975-5327<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas">FAX
205 975-5479<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas"><A
href="mailto:prevelig@uab.edu">prevelig@uab.edu</A><o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10.5pt; FONT-FAMILY: Consolas"><A
href="http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm">http://www.microbio.uab.edu/faculty/prevelige/prevelige-p.htm</A><o:p></o:p></SPAN></P>
<P class=MsoNormal><o:p> </o:p></P></DIV></BODY></HTML>