Re: DAB stability
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | "In C. Kim" <inkim@ingenlabs.com>, Histonet <Histonet@Pathology.swmed.edu> |
Reply-To: | |
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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