H+K+-ATPase Activity Assay Kit

K143

100T (50 samples)

This kit is suitable for detecting the activity of hydrogen-potassium ATPase in animal tissues and cultured cells.

This kit can be stored at 2–8°C or –20°C in the dark for up to 6 months. Please refer to the manual for specific storage conditions of each reagent component.

Note: Reagents must be stored strictly according to the storage conditions specified in the table above; reagents from different test kits should not be mixed. For reagents with small volumes, please centrifuge them before use to ensure an adequate amount is available for measurement.

ATPases can hydrolyze ATP to produce ADP and inorganic phosphate; enzyme activity is determined by measuring the rate at which inorganic phosphate is produced per unit time. In an acidic solution, inorganic phosphate reacts with ammonium molybdate to form an ammonium phosphomolybdate complex, which is then reduced by a reducing agent to yield molybdenum blue; this compound exhibits an absorption peak at 660 nm. The concentration of molybdenum blue is measured to calculate the amount of inorganic phosphate.

When using this kit for sample analysis, the total protein concentration must be determined; the BCA method (Product Code: K001) is recommended.

UV–Vis spectrophotometer (660 nm), double-distilled water, Normal saline (0.9% NaCl)

1.Before testing, balance the reagents at room temperature.

2.Preparation of Reagent 4 Application Solution: Dissolve each vial of powder in 5 mL of double-distilled water upon use; prepare immediately – remaining solution may be stored at temperatures below-20°C for up to one week.

3.Preparation of Reagent 5 Application solution: At the time of use, add 5 mL of double-distilled water to each powder vial, then dissolve at 37 °C; prepare immediately before use; any remaining solution may be stored at 4 °C for up to one week.

4.Preparation of Reagent 7 Application Solution: Add 15 mL of double-distilled water to the Reagent 7 bottle; store at 2–8 °C for 3 months.

5.Preparation of Reagent 8 Application Solution: When needed, add 30 mL of double-distilled water to each bottle for dissolution; store at 2–8 °C away from light for one week.

6. Preparation of Reagent 9 application solution: Dissolve Reagent 9 in 60 mL of double-distilled water; store at 2–8°C for up to 3 months (if a small amount of insoluble powder is present, simply collect the supernatant – this does not affect the results).

7. Preparation of the phosphorus determination reagent: Prepare the reagent according to the volume ratio of H₂O: Reagent 10: Reagent 8: Reagent 9 = 2:1:1:1; prepare it immediately before use. Store the prepared color-developing solution in a light-proof container at 2–8°C for up to 1 day.

8.0.5 μmol/mL Standard Solution Preparation: Prepare by mixing Reagent 11 (10 μmol/mL standard stock solution) with double-distilled water at a volume ratio of 1:19; prepare just before use and store at 2–8°C for up to one week.

9. Preparation of the application solution for control and test tubes:

Note:

① n is the number of samples to be assayed.; to avoid running short of the final test dose, it is necessary to prepare an additional 2 tubes.

② Reagent 6: A precipitate will form at 2–8 °C; simply collect the supernatant, as this does not affect the results.

1.Sample Processing

Tissue sample preparation: The homogenization medium is 0.9% NaCl. After homogenization, the sample was centrifuged at 4 °C using 10,000 × g for 10 minutes; the supernatant obtained was collected for subsequent analysis. A portion of the supernatant was reserved for protein quantification.

Cell samples: The cell samples were subjected to mechanical homogenization or ultrasonication in 0.9% NaCl, then centrifuged at 10,000 × g for 10 minutes at 4°C; the supernatant was collected for analysis. A portion of the supernatant was reserved for protein quantification.

Note: Phosphorus-containing reagents, as well as detergents such as SDS, Tween 20, NP-40, and Triton X-100, should not be used when processing samples.

2.Sample dilution

Prior to the formal assay, 2–3 samples with significant differences in values should be selected, diluted to different concentrations, and used for a preliminary experiment; the dilution ratios for these samples are shown in the table below (for reference only):

Note: The diluent is 0.9% NaCl.

① During the determination process, care should be taken to prevent phosphorus contamination; all containers used for storing reagents and test tubes involved in the detection procedure must be clean; it is preferable to use disposable EP tubes or new glass test tubes.

② The protein concentration of the sample to be tested should not exceed 3 mg/mL.

Definition: One ATPase activity unit is defined as the amount of ATP hydrolyzed by one milligram of tissue protein per hour to produce 1 μmol of inorganic phosphate; this value is expressed as micromoles of phosphorus per milligram of protein per hour (1 μmol Pi/mg prot/hour).

[Note]

A1 :Compare with the OD values of the control tube

A2 :Measure the OD value of the test tube

A3 :Standard pipe OD value

C: Standard solution concentration – 0.5 μmol/mL

*: 4.8 – The sample dilution factor for the reaction system

**: The reaction time is 10 min; enzyme activity is defined as the activity measured over 1 hour of reaction, so it must be multiplied by 6.

Cpr: Protein concentration of the sample to be tested (mg prot/mL)

f: Dilution factor before adding the sample to the detection system

1. This kit is intended solely for research purposes; should it be used for clinical diagnosis or any other purpose, our company shall not be liable for any issues arising therefrom nor shall we bear any legal responsibility.

2. Before conducting the experiment, please carefully read the instruction manual, properly adjust the equipment, and strictly follow the instructions when performing the experiment.

3. During the experiment, please wear lab coats and latex gloves to ensure proper protection.

4. The detection range of the kit does not correspond to the concentration range of the analyte in the sample. If the concentration of the analyte in the sample is too high or too low, please appropriately dilute or concentrate the sample.

5. If the sample being tested does not fall within the list of sample types specified in the instruction manual, it is recommended to first conduct a pilot experiment to validate its detection efficacy.

6. The final experimental results are closely dependent on factors such as the effectiveness of the reagents, the operator's procedures, and the experimental environment. Our company is solely responsible for the kit itself; we are not liable for any sample consumption resulting from the use of the kit. Please carefully estimate the potential sample volume before use and reserve an adequate amount of sample.