Quantification of Western Blot Signals on ECL Film

[Dr Engelbert Buxbaum]

WS wrote:

> I want to quantificate signals on X-Ray films obtained by Western
> Blot / ECL.

If you can show by western that your antibody interacts with only 1
protein in your sample, quantitation is much easier to perform by dot-
rather than western blot.

There are 96-well filtration manifolds commercially available, which can
be used to filter the sample over a high-protein-binding membrane (e.g.
PVDF). Alternatively, there are also 96-well plates available with
filters as bottom. For small samples add PBS or similar to the wells, so
that the sample fills the entire area of the well and you get defined
spot shapes.

After filtration the blot is developed like a western, using
peroxidase-conjugated secondary antibodies. After incubation in a
substrate solution for a few minutes place the membrane in a holder and
that into a 96-well chemoluminescence counter (I used Berthold and was
quite satisfied, but other manufacturers make similar instruments). 

This type of assay is linear over 5-6 orders of magnitude. If you
include calibration standards you get absolute concentrations. And with
96 samples handled in parallel, even large studies can be performed in a
timely and economic manner.