I am using promega ADP-Glo kit. In most of my trials the expected RLU luminescence value is very low even for the standard curve. I talked with the company and the replaced the kit once. But I am still facing the same though I am following protocol in the same way as given. Any idea about this ? Any experience working with ADP-glo?
Hello Mahfuza Marzan,
The intensity and stability of luminescent signal from ADP-Glo kinase assay will depend on factors that affect the rate of luciferase reaction resulting in a change in the intensity of light output and stability of the luminescent signal.
Temperature is one of the factors that affect the rate of this enzymatic assay and thus the light output.
1. Do you thaw all components completely at room temperature before use?
2. Do you mix each of the components thoroughly before use? Mix extensively by vortexing for at least 1 minute.
3. Do you allow the substrate to equilibrate completely to room temperature before use?
4. Do not use thawed assay buffer as well as kinase buffer for more than one day because the components used in the buffer are unstable such as DTT.
Also, the chemical environment may affect the enzymatic rates and thus the luminescence intensity. Always maintain the pH for the kinase buffer in the range of 7–8.
Best.
Hi Malcolm Nobre ,
Thank you so much for your answer in detail. I just want few clarifications please.
When I thaw the provided kinase buffer and I use it, do you think it is fine if I re-freeze it and use it again for next experiment or is it just one time use?
When you mention about the vortex does that mean vortex the the kinase protein with ATP and water before we add it in the well serially? Or vortex the substrate preparation? or only kinase buffers?
Do you follow the promega protocol exactly for buffers preparation or is there any modification that can get better result?
Thank you again. I am really struggling getting high RLU.
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