The Fluoro ATP detection kit utilizes a non-fluorescent detection reagent, which is reduced in the presence ATP and a coupled enzyme reaction to produce its fluorescent analog. There is a linear relationship of ATP concentration to the fluorescent analog concentration. An ATP standard curve is generated to interpolate sample ATP concentrations. The kit can be used in both endpoint and kenitic modes.

Reaction:

1. ATP + Enzyme Coupled Reaction + Non-Fluorescent Detection Reagent  →  Fluorescent Analog + AMP

Detection: Excitation: 530-570nm and Emission at 590-600nm

2. 50µL of sample or ATP Standard

             +

50µL of Enzyme Reaction Cocktail

             ↓  

Incubate 30 – 60 minutes; RT; DARK

Read on Plate Reader: Excitation: 530-570nm

                                       Emission at 590-600nm

Figure 1. ATP vs ADP Standard Curve fitted with linear regression.

Table 1. ATP was spiked into Jurkat cell samples in Substrate Buffer (Part# 3046) with or without 40mM NEM (Part# 7026). % Recovery was determined via linear regression from ATP standard curve. N=3 per sample.