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DETERMINATION OF ATP BY BIOLUMINESCENCE

1.0     PURPOSE

To determine the adenosine 5’-triphosphate (ATP) concentration in nano mole level.

2.0     SCOPE

This test method is suitable for testing ATP in in-vitro assay system or whole cell lysates.

3.0     Background

When luciferin is added to a sample containing luciferase and Adenosine 5’-triphosphate (ATP) ATP, there is an immediate light flash that reaches peak intensity at 0.3–0.5 seconds, and then decays rapidly. To overcome this rapid extinction, Chemediate provides the Luciferase Reporter Assay Kit that ensures optimal sensitivity along with ease of handling. The Luciferase Reporter Assay Kit is suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter.

3.1             Assay principle

Figure 1: ATP dependant bioluminescent reaction catalyzed by luciferase
 
Chemediate offers a bioluminescence assay for quantitative determination of ATP with recombinant firefly luciferase and its substrate D-luciferin (Figure 1). In the assay conditions, the light intensity, with emission maximum ~560 nm, are proportional to the concentrations of ATP and can be conveniently detected by luminometers. This assay is extremely sensitive, and is capable of detect as low as sub-nanomolar levels of pre-existing ATP, or ATP in situ formed in kinetic reacti

1.0     SAFETY

Chemicals may be toxic, corrosive and/or flammable. Proper personal protective equipments should be worn at all time.

2.0     EQUIPMENT

2.1             Analytical balance with accuracy to 0.1 mg.

2.2             Luminometer

2.3             pH Meter

2.4             Calibrated pipetters (10, 20, 200 and 1000 µL) with appropriate tips.

2.5             Disposable Cuvettes, Semi-Micro 1.5 mL Polystyrene, VWR Scientific, 58017-847, 58017-850 or equivalent.

2.6             graduate cylinders, 10 mL, 25 mL, 50 mL

3.0     MATERIALS

3.1             Reagents

3.1.1     D-Luciferin (500 μL, 10 mM) substrate solution with additives

3.1.2     Luciferase, firefly recombinant (50 μL, 1 mg/mL)

3.1.3     Adenosine 5′-triphosphate (ATP) (500 μL, 2 mM solution in TE buffer)

3.1.4     10X Reaction Buffer: 50 mL of Tris-HOAc (500 mM, pH 7.75) with MgSO4(100 mM), EDTA (20 mM) and DTT (40 mM)

4.0     Procedures

4.1             Preparation of 1X assay buffer buffer

4.1.1     Pipette 1.0 mL 10X assay buffer (Section 6.1.4) in a 10 mL graduate cylinder, add milli-Q water to total volume of 10mL.

4.1.2     Label the solution as 1X assay buffer. It is stable for 2 weeks if kept at 4 oC.

4.2             Preparation of standard ATP solutions

Prepare low-concentration ATP standard solutions by diluting the 2 mM ATP solution in dH2O

4.3             Preparation of Substrate/enzyme solutions

4.3.1     The D-luciferin solution (10 mM, 100X) thawed on ice for use and protected from light.  Excess solution can be divided as single use aliquots and stored at -80 oC. 

4.3.2     The Luciferase solution (1mg/mL, assay buffer) should be thawed and kept on ice.  Excess solutions can be divided as single use aliquots and stored at -20 oC. 

5.0     method procedures

5.1             Set the luminometers at light harvesting at all wavelength.  Time course of the light intensity of each assay is recorded for at least 15 minutes. 

5.2             Each sample is tested in triplicate.

5.2.1     Add D-luciferin stock solution (10 mL), Luciferase solution (1mg/mL, 10 mL) and Assay Buffer (960 mL) to a clean cuvette. Add 20 µl of the selected ATP standard solutions prepared in Section 7.2. 

5.2.2     Mix the contents by covering the cuvette with Parafilm and inverting 4-6 times, Insert the cuvette into the sample cell of the flurometer

5.2.3     Measure and record the Luminescence.

5.2.4     Repeat steps 8.3.1-8.3.4 for additional H2O2 standard solution measurements. The fluorescence in blank wells (with exclusion of hydrogen peroxide) is used as a control, and is subtracted from the values for those wells with the H2O2 reactions.

5.2.5     Figure 2 is a representative standard curve of H2O2 measurement. 

5.2.6     Repeat steps 8.2.1-8.2.4 4 with the H2O2 standard solution replaced by the test sample (x µL).  The amount of test sample can be adjusted or diluted to make sure the RFU are in the linear range of the standard H2O2 titration curve (RFU: 200-9000)

5.3             The intensity of the bioluminescence at 10 minutes point

ons.

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