[Arabidopsis] A question about studying plant gene subcellular localization by using eYFP

simonmi1 At post.tau.ac.il via arab-gen%40net.bio.net (by simonmi1 At post.tau.ac.il)
Tue Jan 30 10:13:49 EST 2007


Hello Kam HO

I would advise you from now on to, first of all, try your fusion proteins in
transient transformation. It's much easier, less time consuming and usually if
succesful, it will also work after stable transformation. Regarding your
question the answer is YES. It is possible that in some fusions the protein
will fold corectly and in others it wont. I would advise you 2 things. 1. fuse
your fluorescent protein twice. Once to the C terminus and then a second fusion
to the N terminus. One can never know in advance which will work. 2. It is
possible to construct the fusion protein with a linker between your protein to
the fluorescent one. This may help the protein to fold corectly. You should
look at the literature regarding the recomended linkers.

Sincerely,
Simon Michaeli

Department of Plant Sciences, Tel-Aviv University, Israel

Quoting Chan Kam Ho <h0204121 At gmail.com>:

> Dear all,
>
> I have tried to study the subcellular localization of my gene by fusing it
> in frame with eYFP and also DsRed2. I have placed it under the control of
> CaMV and I have sequenced the constructs and I am sure that the construct is
> correct and there is no misake in the construct. I tried to overexpress the
> construct in transgenic Arabidopsis and since my construct also contains
> CaMV GUS I found that my transgenics are GUS positive but I can't see the
> fluorescence of eYFP/ DsRed2. Is it possible that the fluorescent proteins
> doesn't fold correctly when it is fused with my protein of interest and so I
> can see the fluorescence? does anyone have similar experience to share? What
> can I do now?
>
>
> Thanks in advance
> Kam Ho
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