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Thread: ATP Measurement

  1. #1 ATP Measurement 
    Forum Professor Zwolver's Avatar
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    Hi All

    I am working on standardizing an ATP measurement in soil. However i can only get my (unextracted) standard to have the correct results. My post washing step sample (using it as blanc for the next sample) shows higher results than my actual sample. I am using the ATP/Luciferase reaction to illuminate, and this can be read out by a luminescence meter.

    I suspect the fact i can't find anything is because of the extraction fluid.

    I add 100ul sample to 100ul luciferase mix. The sample should contain ATP from the soil, but also has 670mM Phosphoric acid, 20mM EDTA, 2M Urea, 240mM DMSO and as a binding site blocker i use Adenosine.

    Maybe the pH is too low for luciferase to work? Or the high concentration of urea denatures the protein slightly, or my sample does not contain ATP.. But my actual blanco was higher than my sample, i even tried using demiwater instead of luciferase, and it gave the highest results.

    Any ideas?


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    Forum Professor Zwolver's Avatar
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    I changed the extraction buffer, and i am having results.

    Switched to triton x100 and a pH buffer (sodium diphosphate dibasic), instead of urea and phosphoric acid. Seems like the urea was blocking the excitation of light. Needed to be diluted 10 times with water to see anything, but still repressed. Diluting 100 times would solve the problem, but it would be as high as my blank value.

    Do you know anything about the effect of triton on proteins? I know they use it for some purifications etc. I could use SDS instead of triton, but this was not on stock.


    Growing up, i marveled at star-trek's science, and ignored the perfect society. Now, i try to ignore their science, and marvel at the society.

    Imagine, being able to create matter out of thin air, and not coming up with using drones for boarding hostile ships. Or using drones to defend your own ship. Heck, using drones to block energy attacks, counterattack or for surveillance. Unless, of course, they are nano-machines in your blood, which is a billion times more complex..
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