In article <35C0883D.14AC at ucl.ac.uk>, rmhamso at ucl.ac.uk (Malcolm Ogg) wrote:
> In search of help:
>> I am hoping to begin work in my research group using GFP variants as
> reporter genes in promoter assays.
>> The basic aim will be to co-transfect mammalian cells with one variant
> to normalise for transfection efficiency, and another variant as a
> construct containing the promoter region under examination.
> Thank you for your patience and time
> Yours sincerely
> Malcolm S Ogg
>rmhamso at ucl.ac.uk
Unless the kinetics of your response match the normal (stable)
versions of GFP you should be looking towards using "destabilised"
versions. I've seen destabilised GFP available from Clontech but
I can't comment about BFP, YFP etc.
Also, you may have trouble using two fluorescent proteins
as most GFP forms are so bright they blow away the others. I
realise that BFP forms are getting brighter and brighter but I
don't think they are there yet. The key thing is to check how
good your fluorometer is at distinguishing the two forms you intend
to use - cheaper (filter) machines will probably not be good enough
so you'll need an expensive (monochromator) machine.
My temptation would be to use destabilised GFP as your reporter
and "good 'ol" beta-gal as the control (you can just lyse the cells,
throw in the substrate and read in an ELISA plate reader.
I will be very interested to read the rest of the replies,
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA