Luminescent ATP Detection Assay Kit (ab113849)

We haven’t addressed this question experimentally with the reagents.  The answer is probably.  The lysis reagent is denaturing so bound ATP may be made available to the luciferase. This would be difficult to be definitive and different ATP binding proteins may behave differently.

In theory it should work, but we don’t know for sure as we have not tested it. Therefore, if you are willing to test and publish the data in the form of review on our website then I would like to invite you to our Abtrial programme.

Thank you for your enquiry.

Luminescence does not have multiple wavelengths. In Luminescence mode, the instrument simply measures the emission of visible light from the well. What is key in the measurement is the PMT (photomultiplier time). This acts like the aperture of a camera. The higher the number, the more light gets captured. My suggestion is to accept the default PMT settings of the instrument.

If working with an opaque plate, you may be able to have lower PMTs which will allow for a lower background and greater sensitivity. Either opaque or transparent plates could be used (as shown in the protocol booklet). The advantage of opaque plates is that it allows for a wider dynamic range.

This assay is designed for end-point reading. For development of signal timing, see section C/steps 5 and 6 of the protocol.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact me.

Thank you for contacting us.



The cells do not need to rest or adhere for this assay, you can measure right away.

Any anionic detergent will inhibit the luciferase reaction. What is interesting is that non-ionic detergents have the opposite effect and can in fact enhance the luminescent signal. The following detergents used between 0.5 and 1.5% can enhance the signal = Triton X100, Tween 80, Tween 60, Tween 20. Note that Triton X-100 could potentially rescue the inhibition of SDS.

Other potential components of detergents that could also inhibit the reaction are monovalent metal salts such as KCL at concentrations greater than 1uM, Very high concentrations of divalent metals such as Magnesium (>3mM), Manganese (>2mM) and calcium (>1mM) as they all show a bell shape curve in regards to their effect to the activity of luciferase.

Cell lysis buffers may contain DTAB (0.05 - 2%), BDDABr, sulfobetaine 3-10, , deoxycholate* < 0.05%) and NaF (1-20mM) as ATPase inhibitors, cell-lysing agents like TCA, CTAB or ethanol and enzyme stabilizing agents like THESIT (1-5%), BSA or gelantin.

I hope this helps and if I can assist further, please do not hesitate to contact me.

SDS is a very strong inhibitor of the luciferase reaction. The lowest possible concentration will likely be below 0.05%. We suggest the customer running a standard ATP curve with and w/o SDS to determine empirically the effect.

Theoretically, DTT will not affect luciferase reaction up to a concentration of 50 mM. However, we don’t know what the effect will be on the ATP luminescent kit.

I hope this helps and if I can assist further, please do not hesitate to contact me.

The mammalian cell lysis solution contains 0.1M of alkaline solution. I don't think it will be compatible with protein quantification. Indeed, changes in ATP may be due to unevenness in seeding or low viability. Using multiple replicates per each data point will lower the chances of basing a result in uneven seeding, since low reproducibility could be a sign of uneven seeding from well to well.