Nonrad. ATP synthase assays?

Brian T. Greuel GREUELB1 at JAGUAR.UOFS.EDU
Sun Oct 23 19:06:25 EST 1994


In article <cc37-141094172635 at 132.236.171.168>, cc37 at cornell.edu (CConley) 
writes:

> Hello -- 
>       I'm trying to characterize the activity of ATP synthase from 
> isolated mitochondria. I'd like to use a non-radioactive assay -- I've 
> heard of a luciferase assay, where one adds luciferase, substrates, and 
> ADP to the mitochondria and then measures the fluorescence.  Is this a 
> good assay, what might be problems with it, and are there other assays 
> which I should consider (commercial products)? 
>       Thanks, 
>                                                -- CConley 
>  

I tried the ATP Bioluminescent Assay kit from Sigma this past week in my 
undergraduate Cell Biology lab--it utilizes the luciferin/luciferase system 
you mentioned for measuring ATP concentration.  In my case, it was to measure 
ATP production by isolated chloroplasts, not mitochondria.  This was our 
first attempt at this experiment and we did experience some problems which I 
believe were related to the pH of the reaction mixture.  After illuminating 
the chloroplasts with light for 1 minute, and allowing postillumination ATP 
synthesis to proceed for an additional minute, we stopped the reaction with 
trichloroacetic acid (TCA). Although we diluted the chloroplast suspension 
somewhat with the ATP assay dilution buffer prior to doing the ATP 
bioluminescent assay, we had some problems with inconsistent results among 
the replicate samples.  This may have been due to inconsistent pipetting of 
the TCA (and other reagents) by the undergraduate students so that some 
reactions were inhibited in their light production more than others.  I 
believe the kinks can be worked out, though, and I intend to try it again 
with revisions next year.


Brian Greuel
University of Scranton
greuelb1 at jaguar.uofs.edu   







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