Biochemical measurements of ATP and ADP

Biochemical measurements of ATP and ADP

This protocol enables the quantification of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in whole cell extract of yeast.

Preparation of whole cell extract

1. Preculture yeast cells in 2 ml SC medium using a 16-ml culture tube at 30 °C overnight.
2. Dilute the culture by 40–100-fold with fresh medium.
3. Grow yeast cells to mid-log phase, aiming 2 ×10⁷ cells/ml.
4. Collect the cells in a 1.6-ml microtube by centrifugation for 1 min at 10,000×g.
5. Resuspend the cell pellet in 1 ml fresh SC or 40 mM 2-deoxyglucose medium (for ATP depletion) for 10 min.
6. Separate 50 µl for measurement of cell density, centrifuge for 1 min at 10,000×g.
7. Remove as much supernatant as possible, loosen the cell pellet by tapping or vortex.
8. Add 0.75 ml of 90% acetone, immediately pipetting several times, vortex for a few seconds.
9. In a fume hood, place the tube on a dry block heater, open the lid, incubate for 15 min at 90°C to evaporate the acetone.
10. Centrifuge for 15 seconds at 10,000×g.
11. Further centrifuge the supernatant (< 40 µl) at 20,000×g for 3 min at 4°C.
12. Dilute the supernatant with 450 µl of TE.
13. Aliquot the extract and stored at -80°C until used.

Measurement of cell density

1. Dilute the cell suspension by 10-fold with SC lacking some ingredients essential for cell growth (e.g., histidine, uracil, leucine).
2. Measure optical density at 600 nm (OD₆₀₀) using a spectrophotometer.

Measurement of ATP

1. Prepare 5000 nM ATP by diluting the 5 mM ATP stock included in the measurement kit with deionized water, aliquot and stored at -20°C.
2. Prepare 1000 nM ATP 1.4 ml by diluting the 5000 nM ATP with TE for every measurement.
3. Prepare ATP standards according to Table 1 for every measurement.
4. Prepare ATP premix according to Table 2 for every measurement.
5. Place 10 µl of the ATP standards or experimental samples (whole cell extract) into wells of a 96-well plate (preferably white).
6. Add 90 µl of the ATP premix per well quickly, pipetting 2-3 times.
7. Measure luminescence by a plate reader.
8. Generate a standard curve for ATP concentrations.
9. Calculate the ATP amount in the samples from the standard curve.
10. Normalize the ATP level by the cell number (OD₆₀₀) if necessary.

Table 1. Preparation of ATP standards

Table 2. Preparation of ATP premix

Measurement of ADP

1. Prepare ADP standards according to the manufacture’s instruction note.
2. Place 90 µl of the ATP reagent (mixture of 95 µl assay buffer, 1 µl substrate, 1 µl cosubstrate and 1 µl ATP enzyme) into wells of a 96-well plate (preferably white).
3. Add 10 µl of the ADP standards or experimental samples (whole cell extract) to each well quickly, pipetting 2–3 times.
4. Incubate for 10 min at 22–25°C.
5. Measure luminescence (RLU A) by a plate reader.
6. Add 5 µl of the ADP reagent to each well.
7. Incubate for 2 min at 22–25°C.
8. Measure luminescence (RLU B) by a plate reader.
9. Subtract RLU A from RLU B, and generate a standard curve for ADP concentrations.
10. Calculate the ADP amount in the samples from the standard curve.
11. Normalize the ADP level by the cell number (OD₆₀₀) if necessary.

Materials and Reagents

• Deionized water
• Acetone (FUJIFILM Wako, catalog number: 019-00353)
• 90% (v/v) acetone: mix acetone and deionized water in a 9:1 ratio.
• SC medium, see recipe in ref. 1.
• 2-deoxyglucose medium,see recipe in ref. 1.
• 96-well micro plate, white (ThermoFisher, catalog number: 236105)
• Dithiothreitol (FUJIFILM Wako, catalog number: 045-08974)
• ATP determination kit (Invitrogen)
• EnzyLight ADP assay kit (Funakoshi, EADP-100)
• TE (10 mM Tris-HCl, pH 8.0, and 1 mM ethylenediaminetetraacetic acid)

Equipment

• Tabletop micro refrigerated centrifuge (KUBOTA, model 3520)
• Dry block heater (Labnet International Inc., AccuBlock Digital Dry Bath, model D1200)
• Spectrophotometer (Eppendorf, BioSpectrometer)
• Plate reader (PerkinElmer, Enspire multimode plate reader)

References

1. Takaine, M (2019). QUEEN-based Spatiotemporal ATP Imaging in Budding and Fission Yeast. Bio-protocol 9 (15) e3320. DOI: 10.21769/BioProtoc.3320.