Re: DAB stability

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From:"J. A. Kiernan" <>
To:"In C. Kim" <>, Histonet <>
Date:Sat, 8 May 1999 23:25:20 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Sat, 8 May 1999, In C. Kim wrote:

> I have a question for you.  Diaminobenzidine soluiton (as a substrate for
> peroxidase) is not so stable.
> Do you know what causes decomposition ...

   DAB is easily oxidized to a brown polymer. Normally the
   oxidizing agent is hydrogen peroxide (which is the substrate
   of peroxidase enzymes). Thus, peroxidase accelerates the
   oxidation of DAB by H2O2, a reaction which would occur
   anyway, but more slowly. Oxygen from air also oxidizes DAB.
   If you have old stock, or a bad sample, it will give a
   brown solution. Unoxidized DAB makes a colourless solution.

   Solid DAB must be dissolved first in a small volume of water
   and then diluted with a buffer (usually phosphate or TRIS,
   pH 7.2-7.6). If you try to dissolve it directly in the buffer, 
   the insoluble free base of DAB is formed (loss of protons from
   the DAB4+ ion, from reaction with OH- ions in the slightly
   alkaline buffer). The free base of DAB does not dissolve
   as quickly as the tetrahydrochloride, and needs quite a bit of
   time and agitation to get a clear solution.

   I hope this answers your questions.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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