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Name: Alan S.
Status: Other
Age: 30s
Location: N/A
Country: N/A
Date: N/A 


Question:
The isoelectric point of a protein is that pH at which the protein has no net charge; how does this relate to solubility and the ability of the protein to bind to polystyrene plates in ELISA assays?

Should the pH be adjusted for each protein to optimize binding and does it relate to the isoelectric point?



Replies:
Dear Alan:

As a purely practical guide to this type of question, you might want to consult the following laboratory manual:

The ELISA Guidebook
J.R. Crowther, Humana Press, Totowa, NJ, 2001.
ISBN: 0-89603-728-2

Alan, while I understand your concern, I would not play with the pH in this assay. If you are doing ELISAs, you are presumably screening antibodies that you have raised to a protein. If this is so, the antibodies were almost certainly raised to "native" (i.e., undenatured) protein. If you change the pH while binding the antigen to the plates, you may change the conformation of the protein. This could result in you missing some antibodies that react to the native form.

From a practical standpoint, I do not know of anyone (including my lab) that has had problems with the plastic ELISA plates not binding proteins effectively under normal conditions. If you are raising monoclonals, believe me, this is the least of your concerns. :)

Paul Mahoney, Ph.D.



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