We recently tried the Pierce Renilla-Firefly luciferase kit. As mentioned in the product catalog, I set the detection of signals at 640nm for firefly followed by 525nm for Renilla luciferase. However, by the end of the assay all the read outs show "overflow" of signal for both the wavelengths. Even after diluting the lysates 5-10 times I'm getting the same result.
Following this, I did a spectral scan (between 300-700nm) for samples (after adding the reagent), just the reagent and even blank wells. However, I'm still getting "overflow" for all the data points.
Can any suggest what I'm possibly doing wrong?
Following are the details of my assay protocol:
After 24hr transfection, cells were lysed in 100ul of 1X lysis buffer.
Samples were kept on a rocker for 10-15 mins at RT to ensure proper lysis. The lysates were transferred to eppendorf tubes and centrifuged for 5 mins at 13000rpm. 10ul of sample (in three technical replicates) was added to each well of a black-bottom 96-well plate. 50ul of the working solution was added to each well by the injector and absorbance was captured first at 640nm and then at 525nm.
I've attached 2 exel sheets: one of the samples and second of the spectral scan.
Thanks in advance
Dilute your protein extract samples more in PLB or decrease the gain percentage of the luminious values in the software operating the instrument.
Have you tried using deionized H2O (as a negative control) instead of luciferase, and if so, do you get this "overflow" error as well? If it's a "yes", there's nothing wrong with the reagent - seems like your spectro might be having an issue here ...
You should also have assayed the emission spectra at 613nm & 535nm, not the absorbance spectra. Please determine if your spectro can do this, as a conventional spectro (which only measures absorbance) will not be able to, since the halide lamp source and the detector are placed in the same path, while a fluorometer/luminometer has the detector and the light source aligned at right angles.
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