(by nobody from nospam.not)
Wed Jul 18 06:06:36 EST 2007
Kyle Legate <legatek from hotmail.com> wrote in
news:5g5qfqF3f6eahU3 from mid.individual.net:
> Han wrote:
>> 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.
> It sounds like using luciferase to measure ATP production is a little
> problematic. How about coupling it to a more stable, colourimetric
> reaction? It might be less sensitive, but more reproducible.
Actually it should be quite possible. You just need to know the
limitations and technical procedures. Here is the M&M portion of one
publication in J.Pharmacol.Exp.Ther. 306:238-244, 2003.
Quantification of ATP Release by Luciferin-Luciferase Assay. ATP levels
were measured with a firefly luciferin-luciferase assay-based commercial
kit (ATP bioluminescence assay kit HS II; Roche Diagnostics,
Indianapolis, IN). Samples (50 µl) of each supernatant (synaptosomal
preparations) or coronary effluent (isolated hearts) were pipetted into
appropriate test tubes, placed in a TD-20/20 luminometer (Turner
Designs, Sunnyvale, CA) and processed by autoinjection of 50 µl of
luciferin/luciferase reagent. ATP concentrations were calculated from a
calibration curve constructed the same day using ATP standards included
in the kit. The optimal detection range was between 1010 and 1016 mol
of ATP. The amount of ATP was expressed as picomoles per gram of heart
in ischemia/reperfusion experiments and as femtomoles per milligram of
protein in the experiments with synaptosomes.
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