Westerns; napthol reagent?

Paul Cullen Paul.Cullen at med.monash.edu.au
Thu Nov 9 17:29:47 EST 2000

1)electrophoretic transfer to nylon or equivalent
2)Block with 5% skim milk powder in TBS containing 0.05% Tween 20
3)Add serum and incubate
4)Wash blot in the TBS with 0.05% Tween 20 three times
5)Add horse radish peroxidase conjugated secondary antibody and incubate
6) 3 x wash in TBS with 0.05% Teen 20
7) 1 x wash in TBS no Tween
8) Add chloronapthol substrate and allow color to develop in the dark
for up to 30 min
9) 2 x wash in distilled water to stop the reaction

Chloronapthol solution:
1)Dissolve 15 mg of 4 chloronapthol in 5 ml cold methanol
2)Add 15 microliters of hydrogen peroxide (30% w/v) to 25 ml of TBS
3)mix the napthol solution with the hydrogen peroxide solution just
before use

TBS = Tris buffered saline
Sigma makes 4-chloro-1-napthol (I think the cat# is 604-44-4)

agr at medicine.wisc.edu wrote:
> Hello friends,
> I have been trying to do westerns using ECL Plus reagent from Amersham
> without success and now I would like to try something that I had done
> using a napthol reagen successfully a long time ago; unfortuntately, I
> don't have the method using napthol reagent (don't even remember the
> full name of the compound!).
> Can someone please tell me how to do it using napthol reagent and where
> I can purchase it and other materials that go with it?
> I would very much appreciate an email response
> to "agr at medicine.wisc.edu". Thank you very much in advance.
> Aravind
> Sent via Deja.com http://www.deja.com/
> Before you buy.

Paul Cullen
Bacterial Pathogenesis Research Group
Monash University

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