Luciferase

[Han]

Kyle Legate <legatek from hotmail.com> wrote in
news:5g32olF3f0u12U1 from mid.individual.net: 

> Yvonne Couch wrote:
>> Hi all,
>> 
>> I was to run a luciferase assay to see how much ATP my cells are
>> producing. For this I thought I would take an aliquot of medium from
>> each set of treated cells and use it in the Invitrogen
>> ATP-determination kit which employs luciferase.
>> 
> 
> What makes you think your cells are secreting ATP into the medium?
> Wouldn't it make sense to make a lysate, and therefore concerns about 
> medium are no longer necessary?
> 
Some cells do secrete ATP.  One method for measuring the secretion 
component of platelet aactivation is the measurement of secreted ATP.  
However, plasma contains nucleotidases, and the luciferase does not live 
forever (it's signal degrades over time), so it is necessary to start ATP 
measurement within seconds after adding a stimulus to platelets.  Our lab 
uses Chrono-Log equipment and reagents, so I believe that looking at 
their website may help a little.  In particular, the set of curves on 
this page: <http://chronolog.com/CLNEWS.HTM>.  The equipment is not 
cheap, and really aims at platelet functionalities, rather than just ATP 
measurement.

Making a cell lysate has the "problem" of mixing all cellular 
compartments, both those with ATP and those with nucleotidases and 
phosphatases.  Therefore, again time is of the essence.  The luciferase 
has to see the ATP before the other metabolizing enzymes, AND the 
luciferase signal has to be recorded before either luciferase or other 
enzymes have consumed all the ATP.


-- 
Best regards
Han 
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