Na⁺/K⁺-ATPase Activity Assay Kit (AHM, Micro Method) - BioReagent

Cat. No.: N1505953

GRADE & PURITY BioReagent

Synonyms

Na+K+-ATPase Activity Assay Kit

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Wet ice

Application

ATP Assay, Cell Metabolism, Enzyme activity assay

Overview

ATPase is widely distributed in the biological membrane enzyme systems of plants, animals, microorganisms, and cells. It functions as both a carrier and an enzyme, maintaining the cell membrane potential, regulating osmotic pressure, and providing the driving force for nutrient absorption. It plays an extremely important role in maintaining normal cellular physiological functions and can serve as a key evaluation indicator for cellular energy metabolism and functional integrity.

Detection Principle: Na⁺/K⁺-ATPase decomposes ATP to generate ADP and inorganic phosphorus. Under acidic conditions, inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate, which is then reduced to form a blue phosphomolybdenum complex. This product has a characteristic absorption peak at 660 nm. The activity of Na⁺/K⁺-ATPase can be characterized by the change in absorbance.

Applicable Samples: Serum (plasma), animal and plant tissues, cells, bacteria

N1505953	Component	48T	96T	Storage
N1505953A	Extraction Buffer	60 mL	60 mL×2	2-8℃
N1505953B	Assay Buffer	5 mL	10 mL	2-8℃
N1505953C	Reagent Ⅰ	1EA	1EA	-20℃. Store in the dark.
N1505953D	Reagent Ⅱ	1 mL	2 mL	2-8℃
N1505953E	Reagent Ⅲ	1EA	1EA	2-8℃. Store in the dark.
N1505953F	Reagent Ⅳ	1EA	1EA	2-8℃. Store in the dark.
N1505953G	Reagent Ⅴ	1EA	1EA	2-8℃. Store in the dark.
N1505953H	Reagent Ⅵ	25 mL	25 mL	2-8℃
N1505953I	Standard	5 mL	10 mL	2-8℃

Note: Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.

Required Instruments and Reagents

Microplate reader or visible spectrophotometer (capable of measuring absorbance at 660 nm)

Incubator

96-well plate or micro glass cuvettes, adjustable pipettes and tips

Deionized water

Homogenizer (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Notes
Extraction Buffer	Ready-to-use; Equilibrate to room temperature before use.	Store at 4℃
Assay Buffer	Ready-to-use; Equilibrate to room temperature before use.	Store at 4℃
Working Reagent Ⅰ	Dissolve in 7.2 mL deionized water immediately before use.	Unused reagent can be aliquoted and stored at -20℃ protected from light for 1 week; avoid repeated freeze-thaw cycles.
Reagent Ⅱ	Ready-to-use; Equilibrate to room temperature before use.	Store at 4℃
Working Reagent Ⅲ	Dissolve in 3 mL deionized water immediately before use.	Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
Working Reagent Ⅳ	Dissolve in 25 mL deionized water immediately before use.	Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
Working Reagent Ⅴ	Dissolve in 25 mL deionized water immediately before use.	Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
Reagent Ⅵ	Ready-to-use; Equilibrate to room temperature before use.	Store at 4℃
Standard	Ready-to-use; Equilibrate to room temperature before use.	Store at 4℃
0.5 μmol/mL Standard Phosphorus Application Solution	Dilute the Standard 20-fold: Take 0.1 mL Standard and add 1.9 mL deionized water, mix thoroughly.
Phosphorus Assay Reagent	Prepare by mixing deionized water : Working Reagent Ⅳ : Working Reagent Ⅴ : Reagent Ⅵ = 2 : 1 : 1 : 1. The prepared reagent should be light yellow.	If colorless, the reagent has expired; if blue, indicates phosphorus contamination. Prepare the Phosphorus Assay Reagent freshly before use.

Notes:

(1) Reagent Ⅲ has a pungent odor, Reagent Ⅴ is toxic, and Reagent Ⅵ is corrosive. It is recommended to perform the experiment in a fume hood.

(2) It is best to use new beakers, glass rods, and glass pipettes for reagent preparation, or use disposable plasticware to avoid phosphorus contamination.

2. Sample Preparation

Notes:

(1) Fresh samples are recommended. If not used immediately for experimentation, samples can be stored at -80°C for 1 month. Processed samples must be tested on the same day.

(2) If protein concentration measurement is required, the use of Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

2.1 Plasma, Serum, and other liquid samples

Can be detected directly.

2.2 Animal tissue samples

Homogenize the tissue on ice with Extraction Buffer at a ratio of 0.1 g tissue to 1 mL Buffer. Centrifuge the homogenate at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.

2.3 Bacterial and cell samples

Collect bacteria or cells into a centrifuge tube, centrifuge at 4°C, 12,000 g for 1 min, and discard the supernatant. Add 1 mL of Extraction Buffer per 5×10⁶ bacteria or cells. Ultrasonicate to disrupt the bacteria or cells (power 20% or 200 W, ultrasonic for 3 s, interval 10 s, repeat 30 times). Centrifuge at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.

2.4 Plant samples

Rinse the plant sample with cold PBS to remove surface impurities. Homogenize the tissue on ice with Extraction Buffer at a ratio of 0.1 g tissue to 1 mL Buffer. Centrifuge the homogenate at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.

3. Experimental Steps

3.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 660 nm. For the visible spectrophotometer, zero it with deionized water.

3.2 Enzymatic Reaction (Add the following reagents to EP tubes)

Reagent (μL)	Control Tube	Test Tube
Assay Buffer	65	45
Working Reagent Ⅰ	60	60
Reagent Ⅱ	0	20
Sample	0	100

Mix thoroughly and place in a water bath at 37°C (for mammals) or 25°C (for other species) for exactly 10 minutes.

Working Reagent Ⅲ	25	25
Sample	100	0

Mix thoroughly, centrifuge at 4,000 g, room temperature for 10 minutes, and collect the supernatant.

3.3 Phosphorus Assay (Add the following reagents to a 96-well plate or micro glass cuvettes)

Reagent (μL)	Blank Well	Standard Well	Control Well	Test Well
0.5 μmol/mL Standard P Application Solution	0	20	0	0
Supernatant	0	0	20	20
Deionized water	20	0	0	0
Phosphorus Assay Reagent	200	200	200	200

3.4 Mix thoroughly and let stand at room temperature for 30 minutes. Measure the absorbance at 660 nm and record the values.

Note: A control well is required for each sample. Only one blank well and one standard well are needed. It is recommended to perform a preliminary experiment with 2-3 samples expected to have large differences before the formal experiment. If (A_Test - A_Control) is greater than 0.3, the sample can be further diluted with Extraction Buffer, and the calculated result should be multiplied by the dilution factor, or the amount of sample used for extraction can be reduced.

4. Calculation of Results

Note: We provide two types of calculation formulas: the derived process formula and the simplified formula. They are completely equivalent. It is recommended to use the simplified formula in bold as the final calculation formula.

4.1 Calculation of Na⁺/K⁺-ATPase Activity in Serum (Plasma) and other liquid samples

Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per milliliter of serum (plasma).

Na⁺/K⁺-ATPase activity (U/mL) = C_Standard × (A_Test - A_Control) ÷ (A_Standard - A_Blank) × V_Total ÷ V_Sample ÷ (T ÷ 60) = 7.5 × (A_Test - A_Control) ÷ (A_Standard - A_Blank)

4.2 Calculation of Na⁺/K⁺-ATPase Activity in Tissues, Bacteria, or Cells

(1) Calculation based on protein concentration

Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per milligram of tissue protein.

Na⁺/K⁺-ATPase activity (U/mg) = C_Standard × (A_Test - A_Control) ÷ (A_Standard - A_Blank) × V_Total ÷ (Cpr × V_Sample) ÷ (T ÷ 60) = 7.5 × (A_Test - A_Control) ÷ (A_Standard - A_Blank) ÷ Cpr

(2) Calculation based on sample fresh weight

Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per gram of tissue.

Na⁺/K⁺-ATPase activity (U/g) = C_Standard × (A_Test - A_Control) ÷ (A_Standard - A_Blank) × V_Total ÷ (V_Sample ÷ V_{Sample Total} × W) ÷ (T ÷ 60) = 7.5 × (A_Test - A_Control) ÷ (A_Standard - A_Blank) ÷ W

(3) Calculation based on bacterial or cell density

Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per 10⁴ bacteria or cells.

Na⁺/K⁺-ATPase activity (U/10⁴) = C_Standard × (A_Test - A_Control) ÷ (A_Standard - A_Blank) × V_Total ÷ (V_Sample ÷ V_{Sample Total} × 500) ÷ (T ÷ 60) = 0.015 × (A_Test - A_Control) ÷ (A_Standard - A_Blank)

Parameter Description:

C_Standard: Concentration of the standard tube, 0.5 μmol/mL;

V_Total: Total volume of the enzymatic reaction, 0.25 mL;

V_Sample: Volume of sample added, 0.1 mL;

V_{Sample Total}: Volume of Extraction Buffer added, 1 mL;

T: Reaction time, 10 min;

Cpr: Sample protein concentration, mg/mL;

W: Sample fresh weight, g;

500: Total number of bacteria or cells, 5 million.

Precautions

1. Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.

2. If protein concentration measurement is required, the use of Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

3. Biochemical detection reagents are generally irritating, biologically toxic, etc. For your safety and health, please implement appropriate biosafety protection throughout the experiment, wear lab coats, masks, gloves, head covers, and other safety gear, and perform the experiment in a fume hood or biosafety cabinet.

4. This product is for scientific research use only. It is not intended for clinical diagnosis.

Specifications

Synonyms

Na+K+-ATPase Activity Assay Kit

Specifications & Purity

BioReagent

Stability And Storage

N1505953A, N1505953B, N1505953D, N1505953H, N1505953I: 2-8℃. N1505953C: -20℃. Store in the dark. N1505953E, N1505953F, N1505953G: 2-8℃. Store in the dark.

Storage

Store at 2-8°C, Protected from light, Store at -20°C

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive