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<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial>Peter, I have successfully used both SE and SV for peptides in
that size range at concentrations up to ~15 mg/mL, with absorbance detection.
If this peptide is really associating up to something like a hexamer
you should be able to use 6-channel equilibrium cells, at least for the higher
concentrations, but for samples at low concentrations which are mostly
monomer you'll probably need to do equilibrium in velocity cells so
you can go to 60K. The rotor speed limitations mean you can't really get to very
high sigma values, and that's exactly why you <U>don't</U> want to use
interference optics. However the low sigmas are okay for absorbance data if
(and only if) you don't float the baseline offsets. For the higher
concentrations I think I was using wavelengths somewhere out near 300
nm.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial>I really did the velocity dilution
series mostly to check that the association was fully reversible, but to my
surprise I was able to get a pretty good global fit of the 3 concentrations to a
reversible association model, and the stoichiometry was quite clear (and not
what I expected). I can't recall exactly how high I went in concentration but I
think it was > 5 mg/mL (using 3 mm centerpieces, which is quite
important).</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial>I will warn you though that the non-ideality effects may be
much stronger than you expect (they certainly were for the peptides I ran).
The apparent MW from SE reached a maximum as the concentration went up and then
turned strongly downward, and that maximum MW never really approached the
stoichiometry implied by the best-fit model. In my experience the strong
non-ideality was the biggest drawback to the velocity approach. If you really
need to go higher than 10 mg/mL, the complications of hydrodynamic non-ideality
may be a killer.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial>Good luck and best regards,</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=336031223-09122011><FONT color=#0000ff
size=2 face=Arial>John</FONT></SPAN></DIV>
<DIV dir=ltr align=left>
<HR tabIndex=-1>
</DIV>
<DIV dir=ltr align=left><FONT size=2 face=Tahoma><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 2:57
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-FAMILY: Consolas; FONT-SIZE: 10.5pt">Peter
E. Prevelige Jr.<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Professor<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Dept.
of Microbiology, BBRB 416/6<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Univ.
of Alabama @ Birmingham<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">845
19th St. South<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN
style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Birmingham AL.
35294-2170<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">Phone
205 975-5327<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt">FAX
205 975-5479<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt"><A
href="mailto:prevelig@uab.edu">prevelig@uab.edu</A><o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-FAMILY: Consolas; FONT-SIZE: 10.5pt"><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>