Immunoblot semi-quantitative at best?
Dr E. Buxbaum
EB15 at le.ac.uk
Tue Nov 12 12:30:02 EST 1996
Jeff Brown <jeffb at sccwrp.org> wrote:
>I have read that Western blotting is best used to determine the presence
>or lack of presence of a protein, and should not be used as a
>quantitative tool. The reasoning is that the antibody-antigen
>relationship and antibody-antibody interactions are not a one-to-one
>ratio. Also, homologous antibodies are not always available.
>Is this the consensus? Is ELISA a better quantitative tool?
With westerns you have several problems, which can affect quantitation.
How much of your protein enters the Gel during electrophoresis? How much
of it is mobilised during blotting? How much slips through the blotting
ELISAs tend to be more reliable, but are sometimes difficult to set up
and time consuming.
A reasonable compromise is the dot blot. Filter your protein over a PVDF
(not nitrocellulose) membrane in a 96 well vacuum apparatus (Millipore,
BioRad ect). Develope like a western blott, with HRP coupled detecting
antibody. Use chemoluminescence detection, idealy in a 96 well
chemoluminescence counter (Packard, Berthold). I found a good
concentration-signal correlation over some 3 to 4 orders of magnitude. If
such an instrument is not available, photography of the CL signal and
densitometry may be another option, but the dynamic range will be much
smaller (about 1 order of magnitude, I'd guess). Because of the strong
binding of proteins to PVDF the assay is very sensitive and reliable, and
no long incubation times for binding of the protein to a plate are
needed. Because you are dealing with a membrane rather than a plate, the
washing steps are simple and fast.
A western blot may still be important though, to show that all the signal
comes from a single protein.
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