Na+K+-ATPase Assay Kit/SLBC0065
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Product Detail
Na+K+-ATPase Assay Kit
Note: Take two or three different samples for prediction before test.
Operation Equipment: Spectrophotometer/ Microplate Reader
Cat No: SLBC0065
Size:100T/48S
Components:
Reagent I: Liquid 60 mL×1. Storage at 4℃.
Reagent II: Liquid 4 mL×1. Storage at 4℃.
Reagent III: Powder×2. Storage at -20℃. Dissolve thoroughly with 1 mL of distilled water before use. Prepare when the solution will be used. The rest reagent can be kept at -20℃ for one week.
Reagent IV: Liquid 2 mL×1. Storage at 4℃.
Reagent V: Powder×1. Storage at 4℃. Dissolve thoroughly with 3 mL of distilled water before use. Storage at 4℃.
Reagent VI: Powder×1. Storage at 4℃. Dissolve thoroughly with 5 mL of distilled water before use, can be kept at 4℃ for one week.
Reagent VII: Powder×1. Storage at 4℃. Dissolve thoroughly with 5 mL of distilled water before use, can be kept at 4℃ for one week.
Reagent VIII: Liquid 5 mL×1. Storage at RT.
Standard solution: Liquid 1 mL×1. 10 μmol/mL standard phosphorus liquid, storage at 4℃.
0.5 μmol/mL standard phosphorus working solution: Dilute the 10 μmol/mL standard 20 times to 0.5 μmol/mL standard with distilled water. For example: add 1.9 mL of distilled water to 0.1 mL of standard, mix thoroughly.
Phosphorus content determining reagent: Prepare reagents for determining phosphorus content: make solution as the volume ratio of H2O: Reagent VI: Reagent VII: Reagent VIII =2:1:1:1, which should be light yellow. It lose efficacy if its colour change. Prepare the reagent when it will be used.
Note: It is better to use new beaker, glass rod and glass pipettes, or disposable plastic ware when making reagent to avoid phosphorus pollution.
Product Description:
Na+K+ -ATPase is distributed widely in plants, animals, microorganisms and cells, which catalyzes the hydrolysis of ATP to ADP and inorganic phosphorus. The activity of ATPase can be detected by measuring the amount of inorganic phosphorus.
Reagents and Equipment Required but Not Provided:
Spectrophotometer/ microplate reader, micro glass cuvette/96 well flat-bottom plate, water bath, desk centrifuge, adjustable transferpettor, mortar, ice and distilled water.
I. Sample preparation:
1. Bacteria or cells and tissue:
Bacteria or cells: collecting bacteria or cells into the centrifuge tube, centrifugation and discard supernatant. Suggest add 1 mL of Reagent I to 5 million of bacteria or cells. Use ultrasonication to splitting bacteria and cell (placed on ice, ultrasonic power 20%, ultrasonic 3 seconds, interval 10 seconds, repeat for 30 times). Centrifuge at 8000 ×g for 10 minutes at 4℃ to remove insoluble materials and take the supernatant on ice for test.
Tissue: add 1 mL of Reagent I into 0.1 g of tissue and fully grind on ice. Centrifuge at 8000 ×g for 10minutes at 4℃ to remove insoluble materials and take the supernatant on ice for test.
2. Serum (plasma): detect directly.
II. Determination procedure:
1. Preheat spectrophotometer/ microplate reader for 30 minutes, adjust the wavelength to 660 nm, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:
Reagent (μL) | Contrast tube (C) | Test tube (T) |
Reagent I | 65 | 45 |
Reagent II | 40 | 40 |
Reagent III | 20 | 20 |
Reagent IV | - | 20 |
Sample | - | 100 |
Mix thoroughly, then place the reaction solution in a 37℃ (mammal) or 25℃ (other species) water bath for 10 minutes. | ||
Reagent V | 25 | 25 |
Sample | 100 | - |
Mix thoroughly, centrifuge at 4000 ×g for 10 minutes at room temperature. Take the supernatant. | ||
3. Determination of phosphorus content:
Reagent (μL) | Blank tube (B) | Standard tube (S) | Contrast tube (C) | Test tube (T) |
0.5 μmol/mlstandard phosphorus liquid | - | 20 | - | - |
Supernatant | - | - | 20 | 20 |
Distilled water | 20 | - | - | - |
Phosphorus content determining reagent | 200 | 200 | 200 | 200 |
Mix thoroughly, then place the mix solution in a 40℃ water bath for 10 minutes. Cooling to room temperature and detect the absorbance at 660 nm. The blank tube and standard tube just one or two tubes.
III. Calculation:
1. Serum (plasma):
Unit definition: One unit of enzyme activity is defined as the amount of Na+K+-ATPase catalyzes the hydrolyzation of ATP to produce 1 μmol of inorganic phosphorus in the reaction system per hour every milliliter serum (plasma).
Na+K+-ATPase(U/mL)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷s÷T
=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]
2. Tissue, bacteria or cells
(1) Protein concentration:
Unit definition: One unit of enzyme activity is defined as the amount of Na+K+-ATPase catalyzes the hydrolyzation of ATP to produce 1 μmol of inorganic phosphorus in the reaction system per hour every milligram protein.
NNa+K+-ATPase(U/mg prot)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs×Cpr)÷T
=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]÷Cpr
(2) Sample weight:
Unit definition: One unit of enzyme activity is defined as the amount of Na+K+-ATPase catalyzes the hydrolyzation of ATP to produce 1 μmol of inorganic phosphorus in the reaction system per hour every gram tissue.
Na+K+-ATPase(U/g)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs÷V1×W)÷T
=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]÷W
(3) bacteria or cells
Unit definition: One unit of enzyme activity is defined as the amount of Na+K+-ATPase catalyzes the hydrolyzation of ATP to produce 1 μmol of inorganic phosphorus in the reaction system per hour every 10000 cells or bacteria.
Na+K+-ATPase(U/104 cell )=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs÷V1×500)÷T
=0.015×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]
Cs: Concentrate of standard tube, 0.5 μmol/mL;
Vrv: Total reaction volume,0.25 mL;
Vs: Sample volume, 0.1 mL;
Cpr: Sample protein concentration (mg/mL);
T: Reaction time (min), 1/6 hour;
W: Sample weight, g;
Vl: Volume of reagent I , 1 mL;
500: The amount of bacteria or cells, 5 millions.
Note：
1. As the each sample needs one tube as contrast tube, this kit can detect 48 Na+K+ -ATPase samples in 100 tubes.
2. This method has the characteristics of trace, sensitive and rapid. The test tubes used for determination are phosphate-free strictly.
Experimental examples:
1. Take 0.1 g of mouse heart and add 1 mL Reagent I for ice bath homogenization. After centrifugation at 4℃, 8000g, for 10 min, the supernatant was put on ice, and then the 96 well plate is used to operate according to the determination steps. The results showed that AT= 0.741, AC = 0.509, AS = 0.280, and AB = 0.043
Na+K+ - ATPase activity (U/g mass) = 7.5 × (AT-AC) ÷W= 73.42 U/g mass.
2. Take 0.1 g of barnyard grass and add 1 mL Reagent Ⅰ to homogenize in ice bath. After centrifugation at 4℃, 8000g, for 10 min, the supernatant was put on ice, and then the 96 well plate was used to operate according to the determination steps. The results showed that AT = 0.275, AC = 0.239, AS = 0.280, and AB = 0.043
Na+K+ - ATPase activity (U/g mass) = 7.5×(AT-AC) ÷ W = 11.39 U/g mass.
3. 100 μL of mouse plasma is taken for detection, and 96 well plate is used to measure the enzyme activity: AT= 1.114, AC = 1.054, AS = 0.280, AB = 0.043
Na+K+ - ATPase activity (U/mL) = 7.5 × (AT-AC)/(AS-AB) = 1.90 U/mL.
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References：
[1] Luo L G, MacLean D B. Effects of thyroid hormone on food intake, hypothalamic Na/K ATPase activity and ATP content[J]. Brain research, 2003, 973(2): 233-239.
[2] Cornelius F. Modulation of Na, K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics[J]. Biochemistry, 2001, 40(30): 8842-8851.