I want to know the translocation of AaCdtB toxin using yeast model. We've been trying to tag the toxin with GFP for very long time without any success. What are the critical factors in yeast immunofluorescence experiments, which one should keep in mind?
Hi there,
If I understand well, your goal is to measure toxin translocation through yeast membrane. So if you tag the toxin with GFP, you expect GFP fluorescence signal "to cross yeast membrane". But translocation implies the total unfolding of protein to be translocated and I don't think GFP will be up to unfold on one side of the membrane to refold on the other side because its native structure is stable . I have been trying to do similar experiments with diphteria toxin and nothing came out of it apart that only protein prone to unfolding may cross membrane.
If you intend to transiently express the fusion in yeast and follow GFP fluorescence, you don't need immunofluorescence which start with cell fixation but just live cell fluorescence microscopy because your protein is naturally fluorescent.
Hi Prashant,
There are difficulties with immunofluorescent detections and yeast cells. No doubt about that.
In my lab, we had some success doing it though. I join the paper describing how we proceed (by using several secondary antiboides tagged with a fluorescent dye (not all dyes are equivalent by the way). Please look particularly to Fig . 7 of this paper.
We also succeeded to express various GFPs in yeast in mid 1990s. The best results were obtained with a so-called yeast-GFP, a GFP modified to fit better the yeast cytosol environment.
Best wishes,
Prof Philippe Urban
Hi Prashant,
As I understand you want an alternative assay to the GFP fluorescence as this may not work in your case for reasons already stated here. For the immunofluorescence one of the critical factors is to find an antibody that will specifically recognize a version of your toxin that is not tagged with the GFP. If you have the antibody that is specifically recognizing your toxin then the assay should be relatively easy to perform with the secondary antibody that is conjugated to a fluorophore. Fixing cells is straightforward; perhaps cell digestion may be somewhat tricky. You will also need to perform several washes between subsequent incubations to reduce the background signal.
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