ATPase Activity Assay Kit

K144

96T(40 samples)

0.06-2.75 U/L

0.06 U/L

This kit is intended for the determination of ATPase activity in animal tissue and cell samples.

The kit should be stored at 2-8℃ shading light for 6 months.

ATPase, also known as adenosine triphosphatase, is a crucial high-energy compound in living organisms that functions to maintain cell membrane potential and ionic balance. ATPase catalyzes the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate. The enzyme activity is determined by measuring the amount of inorganic phosphate generated per unit time using a phosphorus determination method.

For the detection of tissue and cell samples with this kit, the total protein concentration must be determined. It is recommended to use our BCA Protein Assay Kit (Catalog No.: K001).

Instruments:

Microplate reader (630-650 nm, optimum wavelength: 640 nm), 37°C incubator, Homogenizer, Refrigerated centrifuge

Reagents:

Double distilled water, Normal saline (0.9% NaCl)

1.Equilibrate all the reagents to room temperature before use.

2.Preparation of Reagent 2 Working Solution: Dissolve the contents of one vial of Reagent 2 in 10 mL of double-distilled water. The working solution can be stored at 2–8°C, protected from light, for 7 days.

3.Preparation of Colorimetric Working Solution: Mix Reagent 4 and Reagent 5 at a volume ratio of 3:1. Incubate at 37°C for 1 hour. Prepare as needed and protect from light. The solution should be used within 10 hours.

4.The preparation of standard curve：

Dilute Reagent 6 with double-distilled water at a volume ratio of 1:99 to prepare a 0.1 mmol/L standard . Prepare as needed and store at 2-8°C.

1.Sample preparation:

Cell samples:

Homogenize the collected cells (10⁶ cells) with 0.9% normal saline (mL) at a ratio of 5:1. Centrifuge at 4°C and 10,000 × g for 10 minutes. Collect the supernatant and keep it on ice for further analysis. Reserve a portion of the supernatant for protein concentration determination.

Tissue samples:

Take fresh tissue blocks and rinse them with 2–8°C PBS (0.01 M, pH 7.4) to remove blood. Blot dry with filter paper and weigh the tissue. Place the tissue into a homogenizer and add 0.9% normal saline at a ratio of weight (g) : volume (mL) = 1 : 9. Homogenize the tissue thoroughly. Centrifuge at 4°C and 10,000 × g for 10 minutes. Collect the supernatant and keep it on ice for further analysis. Reserve a portion of the supernatant for protein determination.

2.Dilution of sample:

Before the official assay, it is recommended to select 2–3 samples with expected large differences and dilute them to several different concentrations for preliminary testing. Based on the results of the preliminary experiment and the linear range of this kit (0.06–2.75 U/L). The recommended dilution factor for different samples is as follows (for reference only):

Note: The diluent is normal saline (0.9% NaCl) . For the dilution of other sample types, please do pretest to confirm the dilution factor.

The standard curve: y = ax + b

Calculation of ATPase Activity in Tissue and Cell Samples:

Definition: One unit of ATPase activity is defined as the amount of enzyme that catalyzes the hydrolysis of substrate to produce 1 μmol of product per minute per gram of protein at 37°C.

ATPase Activity (U/gprot) = (ΔA − b) ÷ a ÷ Cpr ÷ T × f × 1000*

[Note]

y: OD value of standard − OD value of blank (OD value when standard concentration is 0)

x: Concentration of the standard

a: Slope of the standard curve

b: Intercept of the standard curve

ΔA: OD value of sample well− OD value of control well

T: Incubation time, 30 min

Cpr: Protein concentration of tissue sample (gprot/L)

f: Dilution factor of the sample

1000*: 1 mmol/L = 1000 μmol/L

1.All experimental utensils should be rinsed repeatedly (approximately 10 times) to avoid exogenous phosphate contamination.

2.If the absolute OD value exceeds 0.5, the sample should be further diluted and re-assayed.

3.It is recommended to use fresh tissue and viable cells whenever possible.

4.This assay kit is for Research Use Only. We will not response for any arising problems or legal responsibilities causing by using the kit for clinical diagnosis or other purpose.

5.Please read the instructions carefully and adjust the instruments before the experiments. Please follow the instructions strictly during the experiments.

6.Protection methods must be taken by wearing a lab coat and latex gloves during the experiment.

7.If the concentration of substance is not within the detection range exactly, an extra dilution or concentration should be taken for the sample.

8.It is recommended to take a pre-test if your sample is not listed in the instruction book.

9.The experimental results are closely related to the situation of reagents, operations, environment and so on. We will guarantee the quality of the kits only, and NOT be responsible for the sample consumption caused by using the assay kits. It is better to calculate the possible usage of sample and reserve sufficient samples before use.