enhancement of GUS activity by methanol?

[Daniel PERAZZA]

Dear Arabinetters,
10 days ago I asked for a simple method to reduce arabidopsis endogenous
GUS activity during fluorimetric assays using MUG as substrate. Here are
the responses I obtained with a little discussion at the end:

Hwa-Jee, Texas A&M University, wrote
The endogenous GUS activity can be reduced by adding 20% methanol in GUS
reaction buffer. It works in even in X-gluc histochemical staining assay.

James Zhang
Try adding 20% methanol to the assay buffer. I can look up the ref  if you
need it.

Chuck Gasser wrote
Addition of methanol to 20% during staining or fluorimetric assays
dramatically reduces endogenous GUS-like activity without affecting
introduced E. coli GUS activity.  This is usually not necessary in
Arabidopsis as endogenous activity is low.  We have found it to be
essential in staining Brassica, tomato or tobacco tissues.

Toni Schaeffner from Munich, wrote
check the paper by
Kosugi, S. et al. (1990) An improved assay for b-glucuronidase in
transformed cells: methanol almost completely suppresses a putative
endogenous b-glucuronidase activityPlant Sci. 70, 133-140     suppression
of endogenous GUS-like activities by 20% methanol in fluorimetric assay,
gives only 0-15& of the original background in all plant extracts measured.
Addtion of 15-20 % methanol (best added with the substate MUG) enhances GUS
activity by a parallel suppression of endogenous background. Actually I
myself experiences only the enhancement, but I did not have troubles with
end. background.


Therefore, I tried adding 20% methanol and found that the endogenous GUS
activity was totaly abolished, but I didn't noticed any enhancement of the
bacterial GUS enzyme as described by Toni Schaeffner. On the contrary, I
observed a decrease of 3-4 times of the enzymatic activity when compared to
an experiment done without methanol (done at the same moment with the same
protein extracts). Does anybody have a good explaination to this?

Daniel




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        PERAZZA Daniel

address:Laboratoire de Genetique Moleculaire des Plantes - UMR/CNRS 5575
        Universite Joseph Fourier
        BP 53
        F38041 GRENOBLE cedex 09
Tel:    (33) 76-51-48-92
Fax:    (33) 76-51-43-36
Email:  perazza at bio.grenet.fr
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