Application

This kit can be used for detection of ATP content in erythrocyte, culture cells or tissue samples. 

Detection significance

Adenosine triphosphate (ATP) is a nucleoside triphosphate formed exclusively in the mitochondria. It is a high-energy molecule described as the energy currency in all living systems. The chemical energy contained in the phosphate bond of ATP drives most cellular processes. Genetic disorders such as Leber hereditary optic neuropathy, Leigh syndrome, neuropathy, ataxia, and retinitis pigmentosa affect the generation of ATP in the mitochondria.

Detection principle

Creatine Kinase catalyzes adenosine triphosphate and creatine to produce creatine phosphate, then detected by phosphomolybdic acid colorimetry

Experimental instruments

Test tube, Micropipettor, Vortex mixer, water bath, Spectrophotometer (636 nm)

Sample preparation

1. Preparation of erythrocyte sample:

Take whole blood anticoagulated with heparin, and collect underlying erythrocyte. Dilute the sample with double-distilled water for 5 times to prepare 1:4 hemolysis. Mix fully and make hemolysis sample. Take prepared hemolysis samples into glass tube, incubate in boiling water bath for 10 min, then oscillate for 1 min to mix fully. Centrifuge at 4000 r/min for 10 min, then take the supernatant for detection.

2. Tissue sample:

    Weigh the tissue accurately, Adding 9 times of the volume of boiled double distilled water according to the ratio of weight (g): volume (mL) =1:9. Then incubate 10% tissue homogenate in boiling water bath for 10 min, then oscillate for 1 min to mix fully. Centrifuge at 3500 r/min for 10 min, then take the supernatant for detection.

3. Cells samples:

Collect the cells (1×106) and add 0.3~0.5 mL of boiled double distilled water(90-100℃) to prepare homogenate with a homogenizer. Then incubate in boiling water bath for 10 min, then oscillate for 1 min to mix fully. Centrifuge at 3500 r/min for 10 min, then take the supernatant for detection.

Operation steps

Note: cuvettes should be washed with tap water for 10 times, then washed with double-distilled water for 4~5 times, avoid contaminated with phosphorus.

Notes

1. This kit is for research use only.

2. Please progress strictly with operation procedures.

3. The validity of kit is 3 months. It is stable for 1 month after opening.

4. Do not use components from different batches of kit.

5. Avoid phosphorus pollution is the key for assay, it is recommended to use disposable test tubes.

6. The prepared chromogenic agent can be stored at 4℃ for 5 days.

7. It is recommended to prepare 2%~5% tissue homogenate for assay. If ODControl > 1.0, sample should be diluted properly, then take the assay.

8. If the ATP content is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).