Hi, I am performing an enzyme activity assay where Fluorescence is quenched upon successful conversion of substrate to product and the it is an endpoint assay. How do I calculate % activity or % inhibition in this case?
There is no difference in the % inhibition calculation compared with when the fluorescence increases.
% activity = (F - MIN)/(MAX-MIN) x 100
where F is the fluorescence measurement of the sample of interest, MIN is the fluorescence of the fully inhibited sample, and MAX is the fluorescence of the uninhibited sample. Since F < MIN and MAX < MIN, both the numerator and the denominator are negative, so the negatives cancel, leaving a positive value. (Or you could take the absolute values of the numerator and denominator.)
The main issue with using a fluorescence decrease assay instead of increase is that the assay window is usually smaller with a fluorescence decrease.
Adam B Shapiro Thank you so much. This really helped. And yes, I am on the course to make the signal window bigger. :)
1. Quenching: Increased activity can lead to fluorescence quenching.
2. Conformational changes: Active protein conformations may reduce fluorophore accessibility.
3. Binding-induced quenching: Substrate binding can quench fluorescence
1. Enzymes: Substrate binding can quench fluorescence, reducing activity.
2. Protein-protein interactions: Binding can alter protein conformation, reducing fluorescence.
3. Ion channels: Open channel conformation may reduce fluorescence.
Applications
1. Enzyme activity assays
2. Protein-ligand binding studies
3. Ion channel function analysis
Techniques:
1. Fluorescence quenching assays
2. Fluorescence resonance energy transfer (FRET)
3. Fluorescence lifetime measurements
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