Hello,
I performed the ADP-Glo kinase assay. In that case, samples(active protein+substrate+ATP+Kinase buffer+w/wo test compound mixture) were incubated for 45 minutes in 30℃.
In the next experiment, samples were incubated for 55minutes in 30℃.
When the experimental results were analyzed, there was no difference except incubation time, but the overall results were better compared to the previous experiment. I think this could lead to a longer incubation time and a better reaction, so I wonder if it's a reasonable idea.
I look forward to hearing from several researchers. Thank you.
Hello Seeun Jeon
The sensitivity of the ADP-Glo kinase assay is not dependent upon extended incubation. The incubation time is in the range of 40 mins to 1 hour. The maximum sensitivity is achieved once the reaction reaches a steady state. Typically, this may occur within an hour’s time. Also, the time required for the assay to reach steady state may vary, depending upon the cell culture system used.
In your case maybe 55 minutes incubation is more appropriate than 45 minutes. You should also try extending the incubation time to 60 minutes. Just check that out. Beyond 1 hour the signal may remain stable for 3-4 hours.
You may also note that the intensity and stability of the luminescent signal from the ADP-Glo kinase assay will depend on factors that affect the rate of the 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 affects the rate of this enzymatic assay and thus the light output. For consistent results, you may equilibrate assay plates and reagents to room temperature before adding the ADP-Glo kinase assay reagents.
Another factor is the chemical environment which may affect the enzymatic rates and thus luminescence intensity. Always maintain the pH in the range of 7–8 for the kinase buffer. Also, some vehicles used to resuspend the various test compounds, or the reagents used in the kinase reaction buffer may interfere with the luciferase reaction and thus affect the light output of the assay.
Best.
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