[RASMB] Characterizing hetero association via sedimentationequilibrium

[John Philo]

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. 
 
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.
 
John
  _____  

From: rasmb-bounces at list.rasmb.org [mailto:rasmb-bounces at list.rasmb.org] On
Behalf Of Toth IV, Ronald T
Sent: Wednesday, January 22, 2014 1:36 PM
To: rasmb at rasmb.org
Subject: [RASMB] Characterizing hetero association via
sedimentationequilibrium


Hello Members! 

Thank you all for your help in the past! I have a new question!


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?

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?


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.


Thanks again for your help I really appreciate it!

- Ronald Toth
 
Post Doctoral Researcher
Macromolecule and Vaccine Stabilization Center
University of Kansas
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