Kamps's Western Blotting Protocol

ECL or autoradiography?

ECL is an appealing technique because it is quick and very sensitive and does not expose the investigator to radioactivity.The use of radioiodinated protein A to detect bound antibodies is however usually a superior technique.

It is much more quantitative,at least if used in conjunction with a Phosphorimager.

It has a lower background.There is less staining of abundant proteins in the sample,and for reasons that aren't clear,protein A shows much less binding to antibodies present in immunoprecipitates than anti-immunoglobulin antibodies.ECL analysis of immunoprecipitates using a second antibody is almost always blighted by strong staining of the antibody bands and by the staining of abundant proteins in the sample.

Blots developed with iodinated protein A can be re-exposed to film to obtain an optimal image.Alternatively,the Phosphorimager scan can be adjusted to give clear definition of bands.

It is almost as fast as ECL.Despite what was indicated in earlier versions of this protocol,identical incubation and washing intervals can be used.Additionally,a successful blot stained with fresh iodinated protein A can often be visualized in 60 minutes with the Phosphorimager,yielding a quantitative,manipulable image.

Procedure

This procedure was originally optimized by Mark Kamps for analysis with anti-phosphotyrosine antibodies.It is however applicable to almost any form of western blotting with minor modifications.

1)Run an SDS polyacrylamide gel as you would normally.

Before the gel has finished running,be sure that you have started de-gassing the transfer buffer.

Some people soak the gel in transfer buffer for 15 min before assembling the transfer apparatus.Recently,we have been setting up the transfers immediately and things seem to work fine.

We usually use Immobilon membranes for westerns.Although there are suggestions that nitrocellulose may give a lower background than Immoblion in ECL,most people prefer Immobilon even for ECL.

If you are using Immobilon,be sure to wet it first in methanol briefly and then wash away the methanol with transfer buffer.

If you are using a nitrocellulose membrane,soak in transfer buffer prior to assembling the sandwich.

2a)Assemble the transfer folder described below in a large,baking dish containing enough transfer buffer so that all layers are submerged.The orientation of the electrodes is also demonstrated.

(+)POSITIVE ELECTRODE (ANODE)

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