Solarbio Ca++Mg++-ATPase Activity Assay Kit

Ca++Mg++-ATPase Activity Assay Kit

Note: Take two or three different samples for prediction before test.

Operation Equipment: Spectrophotometer

Cat No: BC0960

Size: 50T/24S

Components:

Reagent I: Liquid 30 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. 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. Reagent VI: Powder×1. Storage at 4℃. Dissolve thoroughly with 15 mL of distilled water before use, can be kept at 4℃ for one week.

Reagent VII: Powder×1. Storage at 4℃. Dissolve thoroughly with 15 mL of distilled water before use, can be kept at 4℃ for one week.

Reagent VIII: Liquid 15 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 with distilled water to 0.5 μmol/mL standard. For example: add 1.9 mL of distilled water to 0.1 mL of standard, mix thoroughly.

Phosphorus fixing 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 shows lose efficacy if color is changed, phosphorus pollution if color is change to blue. Prepare the reagent when it will be used.

Note: It is better to use new beakers, glass rods and glass pipettes or disposable plastic ware when making reagent to avoid phosphorus pollution.

Product Description:

Ca++Mg++-ATPase is widely distributed in plants, animals, microorganisms and cells, which catalyzes the hydrolysis of ATP to form ADP and inorganic phosphorus.

Ca++Mg++-ATPase decomposes ATP to produce 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, desk centrifuge, adjustable pipette, water bath, 1 mL glass cuvette, mortar/homogenizer, ice and distilled water.

Procedure:

I. Sample preparation:

1. Bacteria or cells:

Collecting bacteria or cells into a centrifuge tube, centrifugation, and discard supernatant. Suggest add 1mL of Reagent I to 5 million of bacteria or cells. Use ultrasonic to splitting bacteria and cells (placed on ice, ultrasonic power 20%, working time 3 seconds, interval 10 seconds, repeat for 30 times). Centrifuge at 8000 ×g for 10 minutes at 4℃ and take the supernatant on ice before testing.

2. Tissue:

Add 1 mL of Reagent I into 0.1 g of tissue, fully grinding on ice. Centrifuge at 8000 ×g for 10 minutes at 4℃ and take the supernatant on ice before testing.

3. Serum: Directly

II. Determination:

1. Preheat spectrophotometer 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:

3. Determination of phosphorus content, add the following reagents to 1.5 mL EP tube:

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 need one or two tubes.

III. Calculation:

1. Serum:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milliliter of serum.

Ca++Mg++-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

• Protein concentration:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milligram of tissue protein.

Ca++Mg++-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

• Sample weight:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour, every milligram of tissue.

Ca++Mg++-ATPase (U/g weight)=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

• bacteria or cells

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every 10000 cells or bacteria.

Ca++Mg++-ATPase (U/104cell )=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, 0.5 mL; Vs: Sample volume, 0.2 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 cell, 5 million.

Note

1. This kit can detect 24 tubes of Ca++Mg++ -ATPase samples in 50 tubes for each sample need one tube as control.
2. This method has the characteristics of trace, sensitive and rapid. The test tubes used for determination are phosphate-free strictly. Avoiding phosphorus pollution is the key to the success of detection.

Experimental example:

1. Take of pancreas and add 1 mL of Reagent I for ice bath homogenization. After centrifugation at 4℃ for 10 min, the supernatant is put on the ice and operated according to the determination steps. ΔAT = 0.916-0.389=0.527, ΔAS =0.398-0.004=0.394

Ca++Mg++- ATPase activity (U/g mass) = 7.5 × ΔAT ÷ΔAS ÷ W = 100.32 U/g mass.

2. Take 0.1g of willow and add 1 mL of Reagent Ⅰ for ice bath homogenization. After centrifugation at 4℃ for 10 min, the supernatant is put on ice and operated according to the determination steps. The ΔAT=0.137-0.124=0.013, and the ΔAS=398-0.004=0.394

Ca + + Mg + + – ATPase activity (U/g mass) = 7.5×ΔAT  ÷ ΔAS ÷ W = 2.47 U/g mass.

References

[1] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2008, 1777(4): 362-368.

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