Hi there,
I have been putting together an enzyme assay that measures the conversion of one sugar substrate into another sugar substrate of a reductase, with the goal of coupling enzyme 1's activity to NADH fluorescence via enzyme 2. The assay buffer contains NAD+.
The problem I have been having is that in all of my samples, even controls devoid of the substrate sugar, I see an increase (up to doubling) of both fluorescence emission at 450 nm (excitation at 340 nm) and absorbance at 340. I'm using BL21 D3 as my production system. Initially I was using whole cell lysates, which led me to believe the issue was probably related to unused metabolites from the cell swimming around. But I have since then performed a his tag purification and the same pattern is happening. The concentration will rise continuously, usually only for 2 to 3 hours, but sometimes even overnight.
Any theories as to what is going on and what could be done about it would be greatly appreciated.
Hi Cooper,
Have you tried other control reactions?
Without enzyme1,
without enzyme2,
or without NAD+?
I have not tried a reaction without the enzyme at all as of yet, but even then why would I see an increase with no sugar substrate?
It is worth a try though to attempt the experiment without any enzyme, I appreciate the suggestion!
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