Enzyme Active without ATP

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Enzyme Active without ATP

Post by Apoptizzle » Tue Oct 11, 2011 4:05 pm

I'm currently working on an fluorescent assay for a helicase enzyme (in which the helicase unwinds fluorescent DNA, and the fluorescence is quenched once unwound). In other protocols I've seen for the procedure, the reaction is initiated with ATP. I am finding that I see a drop in fluorescence without adding ATP (not as much as with, but a significant amount). The dsDNA is stable as it is, and I get a constant measure of fluorescence with just that - but once I add the helicase, it starts unwinding the DNA (without ATP!).

Now a simple idea I had would be to add ATP and all of the required components to the mixture first, then add the helicase to initiate the reaction. The problem is, almost every single enzyme assay I can find similar to mine, they add the ATP to initiate the reaction. Is there a special reason for them using the ATP to initiate? Or would it be ok to initiate by adding the helicase?

EDIT: Technically I could also initiate by adding the DNA last? In the end, the data you plot is a percent (fluorescent units at time t/initial fluorescent units), so I could maybe use the first data point as F0? Any advice?

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Inland Taipan
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Post by JackBean » Tue Oct 11, 2011 5:02 pm

Hi, is the fluorescence increasing or is there just some background fluorescence?

It doesn't matter much, whether you add last DNA or protein, but usually, the enzyme is in blank reaction, because it can have some absorbance, but since you measure fluorescence, it shouldn't matter much.

Cis or trans? That's what matters.

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