At 12:35 20/04/01 +0100, vous avez écrit:

>Dear All,
>
>Thanks for all the help.  It's cleared up quite a few things.
>
>I had done a pre-immune serum block before my primary antibody and i also
>did an antibody dilution series (sorry - forgot to say).  I didn't think
>of blocking for biotin though - thanks.
>
>I did various antibody-free controls and put some slides straight into
>developer - still brown.
>
>I now suspect the fixative (my unfixed guts worked, but i couldn't use
>them for microscopy) or endogenous peroxidase.
>
>I'm still don't understand the chemical basis of the peroxidase quenching
>step (which is driving me mad).  How can an enzyme be permanently quenched
>by flooding with substrate which i then wash away?  I thought that enzymes
>were recyclable?  How could it destroy the endogenous enzymes but not my
>conjugated enzyme when i develop it? Is the activity non-enzyme based?
>-that would then make sense?

Peroxidase is a somewhat particular enzyme. It catalyzes reactions that 
involve free radical intermediates. Furthermore, its substrate is 
peroxides. Free radicals and peroxides are compounds that don't go well 
with proteins and enzymatic activities. Thus peroxidase "degrades" 
peroxides, but during this process the enzyme denatures progressively until=20
inactive, some kind of a "suicide".
Some techniques to detect peroxidase activities without inactivating the 
enzyme to fast use glucose oxidase to generate the peroxide. Glucose 
oxidase generates the peroxide and is inhibited by the peroxide as soon as=20
its concentration rises. If peroxidase uses this peroxide, you couple both=20
reactions: glucose oxidase produces the peroxide, which is immediately used=20
up by peroxidase. These technique detect peroxidase much slower than 
conventional techniques, but are more sensitive because the actual peroxide=20
concentration are lower and thus less prone to inactivate peroxidase.

If you have a real enzymatic peroxidase activity, you can irreversibly 
inhibit this by using sodium azide. But this treatment does not abolish 
some non-enymatic "pseudoperoxidase" acivities.

>Thanks so muchj for  all of your answers.  I've been getting lost in
>thousands of different protocols for the past few weeks and then suddenly
>got so many answers overnight!
>
>Jenny

Paul
                                                                         -=3D-
                                                                        (o -) O
==========================3D=====oOo==(_)==OOo=============3D================
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.90.24.05.01  Fax. 03.90.24.05.28
========================klosen@neurochem.u-strasbg.fr==================3D=======