Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Here is our experience with Flash tag.  We made Flash-actin, with an  
extra FLAG epitope between the N-terminal (optimized) tetracysteine  
and the actin coding sequence.   Having FLAG epitope served as a  
useful control in preliminary fixed-cell experiments to see that the  
construct was incorporated into F-actin normally.

In trial 1, we labeled the transfected cells with the Invitrogen  
Flash (Lumio green), according to the Invitrogen suggested protocol.   
The result was a high background stain, and no detectable specific  
staining.  We then contacted the Tsien Lab, and they offered some  
hints for improving labeling.  These "hints" were that we use the  
protocols described in the published Tsien et al work, rather than  
Invitrogen.  In the second trial, reducing agents were present during  
labeling and in the washes afterwards.  Also, perhaps just as  
importantly, we noticed that the Lumio Flash reagent is unstable in  
aqueous buffers.  In our first attempt, we had used old DMSO that was  
no longer anhydrous.  Use fresh high-grade DMSO to dilute the FLASH- 
EDT reagent.  The reagent is good if it has a pale pink color; upon  
exposure to water it turns bright orange, and then it no longer works  
as advertised.

The second attempt using good reagent in the present of reducing  
agents produced a very nice specific label.  Background stain was  
still present, but the specific label in transfected cells was  
obviously more intense than the background, which was present equally  
in all cells in the dish.  Label the cells for 30 min or less; longer  
times only increased the background stain.  This is consistent  with  
the high affinity of tetracysteine for Flash and also the instability  
of un-ligated Flash in aqueous buffers.

Flash labeled actin underwent considerable photobleaching during live- 
cell experiments.  We also used Flash-actin for FRAP experiments, but  
here there is a concern that the singlet oxygen generated during the  
bleach is causing damage to the F-actin or to the cell.   Such a  
property would be an advantage for knockout techniques such as CALI.   
We haven't yet tried photoxidation in fixed cells.

Our overall impression is that Flash techniques are a useful addition  
to the fluorescence toolkit, but that GFP and RFP variants are better  
for routine live-cell work.  On that note, however, using Flash  
allowed us to overcome certain abnormal behaviors of GFP-actin that  
are due to GFP steric hindrance, since the Flash-FLAG-actin did not  
cause the same problems as GFP-actin.

Michael J. Schell
Dept. Pharmacology
USUHS
4301 Jones Bridge Rd.
Bethesda, MD 20814

Tel (301) 295-3249
http://www.usuhs.mil/pha/mschell.html
[log in to unmask]