Robert.Roszak at univ-lille1.fr
Thu Feb 2 11:01:25 EST 1995
>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.
>Plant Biotechnology Institute
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.
Equipe de Physiologie et Genetique Moleculaire Vegetales
Bat SN2-3eme etage
59655 Villeneuve d'Ascq Cedex
N. tel. 20 43 65 96
More information about the Arab-gen