Luciferase assay-signal decrease - why this decay? (Jul/31/2007 )

Hi there

I am performing Luciferase assays with an Elk1-Pathdetect Reporter cell line, so the luciferase reporter cassette is stably integrated. My problem is that when I perform the assay with a full 96well plate the signal decrases within the last samples back to low signal. I checked by including a "positive" control in the first wells, which gives high signal and in the last wells, which is then very low, although its the same sample. Delay time is 10 seconds, and counting time 15 seconds. Is the Luciferase assay so unstable that this time differences between first and last samples could explain thhis effect?
Does anyone have same problems or knows where could be the problem?
Thanks a lot

This may indeed be a half-life problem that is inherent in 'flash' type luciferin substrates with high activity, but short signal half-life. Perhaps you could try a "glow" type which gives a lower signal but is stable for ~2hours, and so will vary much less within a plate as a function of time. I like the Dual-Glo kit from promega for my transiently transfected primary cells a with high activity reporter (corrected for Renilla), which is a glow-type. Another reporter I used previously was very lowly expressed in unstimulated cells signal so I had to use a Flash-type and GFP.

If you can't change substrates, you could set up your experiments in several small lots(6 or 8 wells at a time), where you add substrate to only a few wells, then read RLU's, and then move onto the next lot? That is more tedious, but may help preserve your signal between samples if you're forced to use a flash substrate because of low signal.

We use Victor ^ 2 platereader and we set time to 0.1 sec. I think you can reduce reading time to get around this problem.

Hi,
If you're using a 10 second delay before reading each well, then it sounds to me like you're using a plate reader with injectors, correct? If not (i.e. if you're adding the luciferin substrate to all the wells at once) then there's no need for a delay before reading each well.

Anyway, if you are in fact using injectors and you're still seeing a decrease in signal from the first to the last well, then that would be odd, and would suggest degradation of the luciferase protein in your cell lysate over time.

Regardless of the plate reader you are using, there's really no need to read for a full 10 seconds. Sure, it will give you higher numbers, but the signal to noise ratio doesn't change based on reading time, and that's all you really care about.

I've done more luciferase assays in my life than I care to recount... let me know if you have any more questions. I also have some pretty good homemade recipes that I should post at some point...