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Isoelectric Points, Proteins, Solubility
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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Update: June 2012
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