Autofluorescence and GFP fusions

[Sepp D. Kohlwein]

In article <61avdm$325$2 at bignews.shef.ac.uk>, P.Sudbery at shef.ac.uk (P E
Sudbery) wrote:

> We are trying to localise the intracellular location of a 
> S.cerevisiae protein using a GFP fusion. Trouble is our negative 
> controls fluoresce like crazy. We have tried W303 and Berkeley strains 
> and have grown cells on YEPD and minimal media. Washing cells with 
> phosphate buffer helps but doesn't eliminate the problem. We don't 
> think its our microscope because we have also used  GFp fusions in 
> Candida albicans without any problems. Anybody got any ideas?


w303 has the ade2 mutation which results in the accumulation of a red and
highly fluorescent pigment (same excitation wavelength as GFP, and
overlapping emission spectrum) in the vacuole. Make sure to use an ADE+
strain if you have a low GFP signal. 


Sepp D. Kohlwein

-- 
*********************************************************************
Sepp D. Kohlwein, PhD
SFB Biomembrane Research Center                         
Genetics and Molecular Biology Group              
Department of Biochemistry          
Technical University Graz                 phone:  ++43 (316) 873-6456
Petersgasse 12                            fax:    ++43 (316) 873-6952
A 8010 Graz, Austria            e-mail: kohlwein at ftugax.tu-graz.ac.at
*********************************************************************