Dear Kim,
you could try to reduce the background by incubating your sample with
cyanoborohydride or borohydride (you might check PubMed fro protocols) or
alternatively use RFP or EGFP (clontech). from GFP to EGFP it is just a 1
amino acid mutation (F65L IIRC, so you might use site directed
mutagenesis). If you can't change your reporter system, you might use
antibodies against GFP or - if necessary - include an epitope tag like VSV,
HA or myc.
Wolfgang
> Hi my name is Kim Van Beveren I am a PhD student working transformation in
> Radiata Pine. I have just started using the reporter gene GFP but I have had
> some problems with auto fluorescence in the callus material I am working on.
> My controls fluoresce as must as the transform material which has made me
> think that the GFP transformation may not be working and I am in the process
> of confirming it with molecular techniques. To use GFP as a reporter gene
> successfully I have to be able to see the cells that are transform and not
> just
> auto-fluorescence that may be present. If anyone has any experience or
> information on problems with auto-fluorescence vs GFP or the types of GFP
> filters that are used it would be gratefully received.
>> Thanks Kim Van Beveren
>> The School of Forestry
> The University of Melbourne
> Water st, Creswick
> Victoria, 3363
> Telephone: +61 3 53 214122
> facsimile: +61 3 53 214194
> E-mail: ksvb at unimelb.edu.au>
[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
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Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
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