X-gluc problems

[Robert Roszak]

>I am attempting histochemical staining of salt-stressed Arabidopsis
>transformants, using x-gluc to detect GUS activity. Non-stressed plants
>develop low levels of indigo during staining; stressed plants produce some
>indigo in superficial cell layers and develop a red stain in underlying
>cell layers. The latter slowly (over several days) turns blue during
>clearing/fixation using chlorallactophenol. Does anyone know if salts
>(in this case sodium and magnesium sulphates) inhibit oxidative dimerization
>of the indoxyl cleavage product of the reaction? Or if there is perhaps a
>more mundane explanation?
>
>Thanks in advance for any help. I can be reached at the above e-mail address.
>
>Mike Thompson
>Plant Biotechnology Institute
>Saskatoon, Canada.

Dear netter,
I think that such problems are normal. This phenomena is due to enzyme
susceptibility gainst divalent cations. Jefferson have previously
describded such  inhibition. I have an extraction protocol from Lieve
HERMAN and Ann DEPICKER where extraction buffer is

                NaPO4 (pH 7,0)          50 mM
                EDTA                    1 mM
                Triton X100             0,1 %
                2-mercaptoethanol       10 mM

this buffer is also used as reaction buffer. If you use such a buffer, i
think you can increase EDTA concentration so that almost all divalant
cations will be complexed.
I Hope that this explanation will help you.

Sincerely yours.


Robert ROSZAK
Equipe de Physiologie et Genetique Moleculaire Vegetales
Bat SN2-3eme etage
59655 Villeneuve d'Ascq Cedex
France
N. tel. 20 43 65 96