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I measured some enzyme kinetics in a practical course using a substrate-based FRET assay. Unfortunately some of my plots show weird effects. There was always a decrease in signal after 35 minutes. But the curve always regained. During the whole measurement the plate was placed in the reader, and was heated to 37°C.

x axis - time, y axis - fluorescence

My question is: Does someone know how/why this effect occurs?

Thanks for your help!

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    $\begingroup$ Those points are within the error bars of the previous measurements, so perhaps they aren't statistically significant. How many replicates did you do? $\endgroup$
    – MattDMo
    Oct 31, 2021 at 21:25

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Worth considering whether it is an effect of the instrument or the system. What would happen if you preincubated your plate for 10 minutes? Would you have the "dip" after 25 minutes or again 35 minutes? I know our plate reader will want to calibrate its detector at some intervals, perhaps that coincidences with 35 minutes for your combination of variables?

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  • $\begingroup$ In addition to these suggestions, running the samples on a different instrument could be informative. $\endgroup$
    – Galen
    Nov 2, 2021 at 15:34

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