Re: Luciferase detection

hello,

I thought that I read sometime ago that there are a bunch of 
luciferases.  And most of them wont work by flow, but one in particular 
could be used.	Sorry i don't have the reference.  I think i tracked the 
paper down from one of the many times that this came up on this list.
So i guess i dont know if this was any help at all.  :-)
but maybe somebody will know what im talking about and can help me out.

JM


At 04:59 PM 12/18/2006, David Galbraith wrote:
>Hi everyone:
>
>This question does come around regularly!   Has anyone produced a 
>bifunctional GFP-luciferase fusion?  Then we could do a head-to-head 
>comparison of sensitivity using all the available different assay 
>platforms.  In short, the time-of-flight of a cell through a flow 
>cytometer is too rapid to allow meaningful collection of 
>luciferase-mediated photons, despite its exquisite sensitivity over long 
>times of collection of light in imaging modalities.  Going to an 
>antibody-based assay would mean that you lose the advantage of a flow 
>assay in vivo, and probably that you would lose sensitivity (at least 
>relative to GFP or its colleagues).  Howard can probably chime in here as 
>well regarding slow-flow (Happy BD by the way, Howard!).
>
>David
>
>At 12:10 PM 12/15/2006, Gagne Daniele wrote:
>>Hi everyone,
>>
>>I’ve been asked to post this question about luciferase detection:
>>
>>« Luciferase reporter gene expression can be detected and quantified with 
>>very high sensitivity in bioluminescence tests from whole wells 
>>containing cells or organs. However, it is not useful when evaluating 
>>events at the single cell level, due principally to the low emission 
>>intensity of the substrate luciferin. I read the thread of Nan Jiang dec 
>>2002 but was wondering if things had evolved since then. In other words, 
>>I would like to know if someone has found a way of differentiating 
>>luciferase positive and luciferase negative cells by cytometry: either in 
>>microscopy or flow. It could be by the direct detection of activated 
>>luciferin substrate, but also by using antibodies against luciferase. 
>> From my own searches, there are few antibodies (so if someone already 
>>knows of a nice working one for FACS) and we found one protocol 
>>suggesting a 10 min acquisition under the microscope to detect the 
>>activated substrate. Any help would be appreciated,
>>
>>Thank you!
>>
>>
>>Danièle
>>Danièle Gagné
>>Conseillère Technique Cytométrie
>>IRIC, Université de Montréal
>>Bureau 1404, Pavillon Marcelle-Coutu
>>Tél.: (514) 343-6111 x1-8094
>>Fax: (514) 343-7780
>
>
>David W. Galbraith
>Professor of Plant Sciences
>& Professor, Bio5 Institute
>University of Arizona
>Office: 822D Marley Building
>
>Mailing address: Department of Plant Sciences
>University of Arizona
>303 Forbes Building
>P.O. Box 210036
>Tucson Arizona 85721-0036 USA.
>
>Tel: (520) 621-9153
>Fax: (520) 621-7186
>Email: galbraith@arizona.edu
>http://cals.arizona.edu/galbraith
>