Hi everybody,
I would like to ask you advice about b-glucosidase zymograms:
at first, is it better to put substrate (I am using methyl umbelliferyl glucopyranoside) directly into gel, or after running?
I had problems to dissolve substrates both in water and in buffer, is doesn't dissolve just vortexing, I realized that warm water was necessary,hoping that I haven't broken the substrate.
then: is it better to perform a SDS PAGE or a page without SDS ?
and also: How can I see the fluorescence?! I mean.... I put gels on transillumitator but I did not see any fluorescence. That sounds strange cause I do I have b-glucosidase activity in my raw fungal enzymes.
Thanks a lot, Veronica
Hi Veronica,
I have perform zymograms using Congo Red dying, not fluorescence, but I can tell you that I have done SDS PAGE gels for it. After the running of the gel I washed it (proteins renaturalized again) and then I added the Congo red dying and incubated the gel to see the b-glucosidase enzyme activity.
I hope it may help you,
Eva M. Gomez del Pulgar
I would use native PAGE with no SDS nor heating and soak the gel in methyl umbelliferyl glucopyranoside after the run. You may also try a denaturing gel with SDS, but then there is no guarantee that your enzyme(s) will renature properly and recover activity. If you add the substrate in the gel and if your enzyme is active during the run, you will end up with streaks starting at the origin.
dear pierre
thank you!!!! Me too I realized with CMC that putting substrate directly into gels let me see a coudy smear and I can't appreciate exactly where activity was, so I should try incubating gel into substrate after EF run. I tried SDS page of a non heated sample and I saw fluorescence activity.
YES! I must try Native PAGE without SDS , thanks a lot for your advices
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