Horseradish peroxidase method

Henk Veldman H.Veldman-2 at
Fri Jun 14 02:08:19 EST 2002

Harald Ottenhof schreef:

> I have recently started to use a secondary antibody with an immunocoupled
> peroxidase (anti-chicken IgG, Sigma A9046) in my westerns. However, I do not
> see any bands developing when I add the substrate. Suspicious that there
> were even no background bands I added a bit of the secondary antibody
> directly to some of the substrate in anticipation of an obvious colour
> change - yet nothing happens?!
>  I know the secondary antibody is fine because another lab is using it for
> their Elisa's with success. All other reagents such as hydrogen peroxide and
> 4-chloro-1-naphthol are fresh as they were recently purchased from Sigma.
> Being fairly inexperienced with the horseradish peroxidase system I'm hoping
> someone can possibly point out an *obvious* problem. Such as, is the
> 4-chloro-1-naphthol a suitable substrate or should I be using the
> diaminobenzidine tetrahydrochloride instead?

When we are not using ECL we use the same peroxidase substrate as the one we use
for immunohistochemistry: DAB with Ni and Co intensification, producing a dark
gray to black reaction product; see: Adams, J Histochem Cytochem 29, 775-780,

There are two common problems with peroxidase detection that people often run

 - The hydrogen peroxide stock has deteriorated. The stuff is unstable and once
it starts decomposing this can proceed quite quickly. You can check on the
concentration by accurately measuring the specific weight (30% should read

 - Never use sodium azide as a preservative in any solution that contacts the
peroxidase, since it will inactivate the enzyme. You could use merthiolate
(Thiomersal) instead.

Hope this helps


H. Veldman
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)

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