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<DIV dir=ltr align=left><SPAN class=920125015-23012014><FONT color=#0000ff
size=2 face=Arial>Ron, it definitely helps with both the AUC experiment design,
and later with the data analysis, if you can assess the preferred
binding stoichiometry (or at least the molar ratio) first. Titration calorimetry
should give you the latter (even if the Kd is too low to measure), and if the
affinity is strong enough for the complex to stay together on an SEC column then
SEC-MALS will give you the stoichiometry. </FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=920125015-23012014><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=920125015-23012014><FONT color=#0000ff
size=2 face=Arial>Since your group mostly works with protein pharmaceuticals I
suspect this ligand may bind very strongly. Note that if the Kd is below a few
nanomolar you may only be able to put an upper bound on Kd by
AUC.</FONT></SPAN></DIV>
<DIV dir=ltr align=left><SPAN class=920125015-23012014><FONT color=#0000ff
size=2 face=Arial></FONT></SPAN> </DIV>
<DIV dir=ltr align=left><SPAN class=920125015-23012014><FONT color=#0000ff
size=2 face=Arial>John</FONT></SPAN></DIV>
<DIV lang=en-us class=OutlookMessageHeader dir=ltr align=left>
<HR tabIndex=-1>
<FONT size=2 face=Tahoma><B>From:</B> rasmb-bounces@list.rasmb.org
[mailto:rasmb-bounces@list.rasmb.org] <B>On Behalf Of </B>Toth IV, Ronald
T<BR><B>Sent:</B> Wednesday, January 22, 2014 1:36 PM<BR><B>To:</B>
rasmb@rasmb.org<BR><B>Subject:</B> [RASMB] Characterizing hetero association via
sedimentationequilibrium<BR></FONT><BR></DIV>
<DIV></DIV>
<DIV
style="FONT-SIZE: 10pt; FONT-FAMILY: Tahoma; COLOR: #000000; DIRECTION: ltr">Hello
Members!
<DIV><BR></DIV>
<DIV>Thank you all for your help in the past! I have a new question!<BR>
<DIV><BR></DIV>
<DIV>Given that the standard procedure for characterizing a self association
with sedimentation equilibrium is to sediment three different concentrations at
three different rotor speeds, my question is this: Is there a standard procedure
for characterizing a hetero association?</DIV>
<DIV><BR></DIV>
<DIV><SPAN style="FONT-SIZE: 10pt">Should I sediment three samples at different
total protein concentration while keeping the molar ratio of antibody to ligand
constant? Should I sediment three different molar ratios of antibody to
ligand?</SPAN></DIV>
<DIV><SPAN style="FONT-SIZE: 10pt"><BR></SPAN></DIV>
<DIV><SPAN style="FONT-SIZE: 10pt">Some further information: We have an
antibody-ligand system, and the ligand is 20 kDa. The ligand binds in the Fc
region, so it's not necessarily limited to a divalent interaction.</SPAN></DIV>
<DIV><SPAN style="FONT-SIZE: 10pt"><BR></SPAN></DIV>
<DIV><SPAN style="FONT-SIZE: 10pt">Thanks again for your help I really
appreciate it!</SPAN></DIV>
<DIV>
<DIV><BR>
<DIV style="FONT-SIZE: 13px; FONT-FAMILY: Tahoma">
<DIV style="FONT-SIZE: 13px; FONT-FAMILY: Tahoma">
<DIV><FONT size=2 face="Times New Roman"><SPAN style="FONT-SIZE: 16px">
<DIV style="MARGIN: 0px"><FONT size=3 face="Times New Roman,serif"><SPAN
style="FONT-SIZE: 12pt"><FONT size=2 face=Tahoma,sans-serif><SPAN
style="FONT-SIZE: 10pt">- Ronald
Toth</SPAN></FONT></SPAN></FONT></DIV></SPAN></FONT></DIV>
<DIV><FONT size=2 face="Times New Roman"><SPAN style="FONT-SIZE: 16px">
<DIV style="MARGIN: 0px"><FONT size=3 face="Times New Roman,serif"><SPAN
style="FONT-SIZE: 12pt"><FONT size=2 face=Tahoma,sans-serif><SPAN
style="FONT-SIZE: 10pt"></SPAN></FONT></SPAN></FONT> </DIV></SPAN></FONT></DIV>
<DIV><FONT size=2 face="Times New Roman"><SPAN style="FONT-SIZE: 16px">
<DIV style="MARGIN: 0px"><FONT size=3 face="Times New Roman,serif"><SPAN
style="FONT-SIZE: 12pt"><FONT size=2 face=Tahoma,sans-serif><SPAN
style="FONT-SIZE: 10pt">Post Doctoral
Researcher</SPAN></FONT></SPAN></FONT></DIV></SPAN></FONT></DIV>
<DIV><FONT size=2 face="Times New Roman"><SPAN style="FONT-SIZE: 16px">
<DIV style="MARGIN: 0px"><FONT size=3 face="Times New Roman,serif"><SPAN
style="FONT-SIZE: 12pt"><FONT size=2 face=Tahoma,sans-serif><SPAN
style="FONT-SIZE: 10pt">Macromolecule and Vaccine Stabilization
Center</SPAN></FONT></SPAN></FONT></DIV></SPAN></FONT></DIV>
<DIV><FONT size=2 face="Times New Roman"><SPAN style="FONT-SIZE: 16px">
<DIV style="MARGIN: 0px"><FONT size=3 face="Times New Roman,serif"><SPAN
style="FONT-SIZE: 12pt"><FONT size=2 face=Tahoma,sans-serif><SPAN
style="FONT-SIZE: 10pt">University of
Kansas</SPAN></FONT></SPAN></FONT></DIV></SPAN></FONT></DIV></DIV></DIV></DIV></DIV></DIV></DIV></BODY></HTML>