I am measuring enzyme kinetics and see the spike at early time points but later is dropping significantly and behave normally. Any thoughts would be appreciated. The file is attached.
I am measuring NADPH production using Abcam Kit for Glucose-6-Phosphate dehydrogenase and the fluorescent probe is designed to bind NADPH. NADP is added to each reaction and kinetics of the enzyme is measured. My guess is that I have something fluorescent in cultured cells , but not blood that is converted to something non-fluorescent overtime.
Thanks a lot everyone
@Adam: cellular NADPH would actually give a burst in fluorescence and then linear response would occur...
Thanks Adam and Dominique.
I probably need to talk to the company tech support too. My concern is that if it is an endogenous NADPH then I should not see decrease of the signal over time. All NADPH that is present in the tube is reacting with the fluorescent probe and the fluorescence accumulating over time. It is like qPCR signal. Probably it is something else that fluoresce in the same part of the spectrum but eventually this compound is disappearing. This is what I think but I need a second opinion. Thanks everyone for helping me. Enzymology is a a new area for me.
If working with suitable NADP concentration you could try to monitor NADPH formation at 340nm and see what happens...
I am not familiar with the kit you're using, but if possible at all I would try measuring the kinetics in the absence of added NADP. Would you still be seeing the spike? Would you see nothing afterwards? Addressing these issues maybe can help you sort out this artifact. Also, testing the reaction using different amounts of cells could help.
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