Horseradish peroxidase method

Harald Ottenhof hho21 at
Thu Jun 13 23:22:09 EST 2002

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?

Any advice appreciated!

My protocol for developing the blot is shown below for reference:
1. Dissolve 300 mg of 4-chloro-1-naphthol (CalBioChem) in 10 ml of methanol.
2. Add 100 ul of this solution with stirring to 10 ml of 50 mM Tris, pH 7.6.
precipitate is then removed by filtering through Whatmann No. 1 filter
3. Add 100 ul of 3% H2O2.
4. Filtered again to remove any white precipitate.
5. Apply to immunoblot and develop at room temperature.

More information about the Methods mailing list