ATP Assay Kit (Spectrophotometry)

ATP reacts with creatine in the presence of creatine kinase to produce phosphocreatine. The generated phosphocreatine can be quantified at 700 nm using the phosphomolybdate colorimetric method, which reflects the ATP content.

Taking 50T/24S packing for example:

Note: Perform a pretest with 2–3 representative samples before formal measurement.

1. Preparation of Lab Instruments

Spectrophotometer or microplate reader、Water bath、Adjustable micropipettes、1 mL glass cuvettes、Mortar and pestle、Distilled water.

2. ATP Extraction

From serum or plasma:

Mix sample with distilled water at a 1:5–10 ratio (e.g., 0.1 mL serum + 1 mL water), homogenize on ice, heat at 100 °C for 5 min, then centrifuge at 8000 g, 4 °C for 15 min. Collect the supernatant for assay.

From tissues:

Mix tissue with distilled water at a 1:5–10 ratio (e.g., 0.1 g tissue + 1 mL water), homogenize on ice, heat at 100 °C for 5 min, then centrifuge at 8000 g, 4 °C for 15 min. Collect the supernatant.

From cells or bacteria:

Collect cells or bacteria by centrifugation, discard supernatant. Add distilled water at 500–1000:1 ratio relative to cell/bacterial number (e.g., 5×10^6 cells in 1 mL water), lyse by sonication on ice (1 min, 20% amplitude or 200 W, 2s pulse / 1s pause), then centrifuge at 8000 g, 4 °C for 15 min. Collect the supernatant.

3. Assay Procedure

1)Preheat spectrophotometer or microplate reader for at least 30 min, set wavelength to 700 nm, and zero with distilled water.

2)Prepare the color reagent immediately before use: mix CB0032S-D and CB0032S-E at 1:5 ratio according to the number of samples (0.87 mL per sample).

3)Sample measurement (add reagents in EP tubes):

Note: Typically, one or two replicates for blank and standard tubes are sufficient.

4. Calculation

1)Serum or plasma:

ATP (µmol/mL)=[Cstd × (Att−Ablk) ÷ (Astd−Ablk)×V1]÷ (V3×V1÷V2) =5×( Att−Ablk) ÷(Astd−Ablk)

2)Tissue, bacteria, or cells:

By protein concentration:

ATP (µmol/mg prot)=[Cstd × (Att-Ablk)÷(Astd-Ablk)×V1]÷(V1÷Cpr)=0.5×(Att-Ablk)÷(Astd-Ablk)÷Cpr

By fresh weight:

ATP (µmol/g tissue)=[Cstd × (Att-Ablk)÷(Astd-Ablk)×V1]÷(W×V1÷V2)=0.5×(Att-Ablk)÷(Astd-Ablk)÷W

By cell/bacterial number (per 5×10^6 cells):

ATP (µmol/10^4 cell)=[Cstd × (Att-Ablk)÷(Astd-Ablk)×V1]÷(500×V1÷V2)=0.001×(Att-Ablk)÷(Astd-Ablk)

Note:

Cstd: standard solution concentration (0.5 µmol/mL)

Att: A test tube

Ablk: A blank tube

Astd: A standard tube

V1: sample volume in reaction (0.03 mL)

V2: extraction volume (1 mL)

V3: serum/plasma volume (0.1 mL)

Cpr: protein concentration (mg/mL)

W: sample weight (g)

500: total number of cell or bacterial (5million).

1.Detection limit: 10 nmol/mL or 10 nmol/g fresh weight or 0.1 nmol/mg prot.

2.For protein quantification, it is recommended to use TargetMol BCA Protein Quantification Kit (C0050).

3.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

4.Please wear a lab coat and disposable gloves.

• CB0032S-Instruction Manual(272 KB)