Determination of on-rate and off-rate

[Pita Enriquez Harris]

> I am working with a small peptide (MW < 500) that binds to a specific 
> receptor. I would like to determine both the on-rate and off-rate of the 
> peptide with the receptor.  Both the purified receptor as well as cell lines 
> that express the receptor are available.  I would appreciate suggestions or 
> references on procedures that can be used to determine on- and off-rates.
> 
> Thanks in advance.
> 
> George A. Heavner
> Senior Director, Peptide R&D
> Centocor, Inc.

If you have access to a machine which measures surface plasmon resonance, 
such as the Biosensor BIAcore or Fisons IASys, you can do on-off rates 
rather easily. You need to have some soluble receptor protein which you 
can then couple to the sensor chip.  You then add your ligand in a 
buffer; this is either flowed over the chip in BIAcore or else simply 
added to the chamber which holds the chip. The machine shows you 
association and dissociation in real time.  The software enables you to 
calculate on-off rates with ease. It is necessary to have a model for the 
association/dissociation; if you have no idea at all about stoichiometry 
it can be tricky to fit the data to the models with any accuracy. But if 
you have plenty of soluble receptor and ligand this method can be very 
useful.

Pita

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Dr. Pita Enriquez Harris           "Waste no more time arguing what a
Nuffield Department of Medicine           good man should be.  Be one."
Oxford Radcliffe Hospital                                  Marcus Aurelius
http://sable.ox.ac.uk/~enriquez
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