Westerns; napthol reagent?

agr at medicine.wisc.edu agr at medicine.wisc.edu
Fri Nov 10 10:56:28 EST 2000


Thank you very much, Paul, for the precise method! I know that this
method is less sensitive than ECL methods, but by the same token, it
could help reduce non-specific staining that I have been getting.

I wonder whether I can use PBS, instead of TBS, since all my reagents
including the antibodies are in PBS.

Thanks again. Comments would be very much appreciated.

Aravind


In article <3A0B255B.5956ED28 at med.monash.edu.au>,
  Paul Cullen <Paul.Cullen at med.monash.edu.au> wrote:
> 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)
>


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