[RASMB] Characterizing hetero association via sedimentation equilibrium

[Laue, Thomas]

Hi Ron-
In addition to sedimenting different total concentrations at different rotor speeds, you need to sediment different molar ratios of the components (at different rotor speeds). Also, you will need to sediment each component alone in order to determine whether self-association needs to be considered.
All of this should be done in conjunction with sedimentation velocity analysis. The biggest hurdle in this work typically is determining the stoichiometries of assembly. Sometimes it is impossible to establish the stoichiometries uniquely (several will fit the data equally well). This may be an appropriate project for the genetic algorithm approach available in UltraScan, and definitely will require the use of programs that combine equilbrium and velocity analyses.
Best wishes,
Tom

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From: rasmb-bounces at list.rasmb.org [rasmb-bounces at list.rasmb.org] on behalf of Toth IV, Ronald T [Toth.Ronald at ku.edu]
Sent: Wednesday, January 22, 2014 4:35 PM
To: rasmb at rasmb.org
Subject: [RASMB] Characterizing hetero association via sedimentation equilibrium

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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