[western blotting] reproducible detection in western blotting

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum At hotmail.com)
Sat Dec 2 05:03:25 EST 2006


Lechu wrote:

> What you would reccommed as an alternative to ECL systems + standard
> autoradiography film? I start to find  film developing pretty boring

Detection of HRP can be done with diaminobenzidine directly on the blot,
it gives a brown precipitate. If you include Ni or Co  in the reagent,
you get black bands from precipitated metal instead, sensitivity is
higher and the blots do not fade when stored even for years in the
notebook. I use 0.5% DAB, 0.01% H2O2 und 1.75% NiSO4 in 50 mM Tris-HCl
pH 7.5. Just watch the blot develop and stop with a brief wash in
distilled water once you are satisfied with the intensity. Sensitivity
is however about 1 order of magnitude lower than chemoluminescence.

BTW: DAB is a suspected carcinogen and should be handled with care. It
is best to make a 10 times stock solution by adding water directly to
the bottle, assuming that the manufacturer has sold you the correct
weight. Do _not_ wear gloves when you do that, otherwise the fine powder
blows into your face electrostatically. The solution is stable for
months in aliquots at -20 degrees.




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