GFP variants

John Runions cjr41 at cam.ac.uk
Mon Mar 19 13:04:04 EST 2001


Hi Madelaine,

For thew time being stick with ECFP and EYFP if you want to label two
proteins within a cell.  These fluorescent proteins are easily
distinguished
even if your proteins of intertest are situated fairly closely to each
other.

I have done some experiments with ECFP, GFP, and EYFP on glass beads on
the
same slide and it is possible to distinguish three colours if sequential

scanning is used but the results would be difficult to interpret unless
your
proteins were differentially localized because of the cross-talk between

ECFP-GFP and GFP-EYFP (I can send a tif image of this to anyone who
might be intertested, the list won't allow attachements).

We have tried dsRED and we get fluorescence but the distribution is not
as
expected.  Several people have reported this and we suspect that it is
because the protein is an obligate tetramer and is clumping up in the
cell.
Certainly the spectrums of GFP and dsRED make them a good contrasted
pair
and we hope that the dsRED problem is worked out soon.  Consider however

that emmision of RFP may overlap with chlorophyll autofluorescence (I
think
it's a little oranger so probably not a big deal).

Good luck, John

Madeleine Rashbrooke wrote:

>  Dear All,
>
>  I am in the process of planning a project involving the construction
of a
>  fusion of a fluorescent protein to my protein of interest. In order to

>  leave
>  open the possibility of double labelling with existing GFP constructs,
I
>  need to use a non green variant. I have a couple of questions that I
was
>  hoping somebody would be able to help me with, based on their own
>  experience
>  with fluorescent tags in arabidopsis.
>
>  Firstly, what is the best source of arabidopsis-optimised fluorescent
>  protein coding sequences? Clontech have an extensive collection
>  advertised,
>  and claim that they work well in plants, but has anyone first hand
>  experience of using these vectors for arabidopsis projects?
>
>  My other question relates to pairs of fluorescent protein colours - I
>  gather that cyan and yellow are the best combination, especially if
one
>  wishes to try FRET, but has anybody had any success with double
labelling
>  of
>  GFP and the new "red" variants such as Clontech's dsRED? Alternatively
is
>  it
>  possible to separate the spectra of cyan and green, and green and
yellow,
>  sufficiently to label two different proteins in the same cell with
these
>  pairs?
>
>  I promise to post any (or all) replies back to the newsgroup - I
suspect
>  it's not just me who's confused....
>
>  Thank you very much in advance,
>
>  Madeleine
>
>  Madeleine Rashbrooke
>  PhD student, Wasteneys lab
>  Plant Cell Biology Group
>  Research School of Biological Sciences
>  The Australian National University
>  GPO Box 475
>  Canberra ACT 2601
>  Tel: +61-2-6125 5561
>  Fax: +61-2-6125 4331
>  Email: rashbrooke at rsbs.anu.edu.au
>
>  ---

--
C. John Runions, Ph. D.
Department of Plant Sciences
University of Cambridge
Downing St.
Cambridge
UK   CB2 3EA

email: runions at plantsci.cam.ac.uk
phone: (01223) 766 545

http://www.plantsci.cam.ac.uk/Haseloff/JohnRunions/Home.html


--
C. John Runions, Ph. D.
Department of Plant Sciences
University of Cambridge
Downing St.
Cambridge
UK   CB2 3EA

email: runions at plantsci.cam.ac.uk
phone: (01223) 766 545

http://www.plantsci.cam.ac.uk/Haseloff/JohnRunions/Home.html


---




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