Solarbio ca ++ mg ++-komplet za ispitivanje aktivnosti ATPase

Ca++Mg++-ATPase Activity Assay Kit

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar

Mačka ne: BC0960

Veličina: 50T/24s

Komponente:

Reagens i: Liquid 30 ml × 1. Skladištenje na 4 ℃.

Reagens II: Liquid 4 ml × 1. Skladištenje na 4 ℃.

Reagens iii: Powder×2. Skladištenje na -20 ℃. Dissolve thoroughly with 1 ML destilirane vode prije upotrebe. The rest reagent can be kept at -20℃ for one week.

Reagens IV: Liquid 2 ml × 1. Skladištenje na 4 ℃.

Reagens v: Prah × 1. Skladištenje na 4 ℃. Dissolve thoroughly with 3 ML destilirane vode prije upotrebe. Reagent VI: Prah × 1. Skladištenje na 4 ℃. Dissolve thoroughly with 15 ML destilirane vode prije upotrebe, can be kept at 4℃ for one week.

Reagent VII: Prah × 1. Skladištenje na 4 ℃. Dissolve thoroughly with 15 ML destilirane vode prije upotrebe, 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: dodati 1.9 ml destilirane vode do 0.1 mL of standard, Temeljito pomiješajte.

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.

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

Opis proizvoda:

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.

Potrebni reagensi i oprema, ali nisu pruženi:

Spektrofotometar, stolna centrifuga, Podesiva pipeta, vodena kupelji, 1 ML staklena kiveta, minobacač/homogenizator, led i destilirana voda.

 

Postupak:

Ja. Priprema uzorka:

1. Bakterije ili stanice:

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 (stavljen na led, ultrasonic power 20%, working time 3 sekundi, interval 10 sekundi, ponoviti za 30 vrijeme). Centrifuga na 8000 × g za 10 minutes at 4℃ and take the supernatant on ice before testing.

2. Tkivo:

Dodati 1 mL of Reagent I into 0.1 g tkiva, fully grinding on ice. Centrifuga na 8000 × g za 10 minutes at 4℃ and take the supernatant on ice before testing.

3. Serum: Directly

Ii. Odlučnost:

1. Predgrijan spektrofotometar za 30 minuta, Podesite valnu duljinu na 660 NM, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:

Reagens (μL)	Kontrola (C)	Epruveta (T)
Reagens i	130	90
Reagens II	80	80
Reagens iii	40	40
Reagens IV		40
Uzorak		200
Temeljito promiješajte, then place the reaction solution in a 37℃ (sisavac) ili 25 ℃ (Ostale vrste) vodena kupka za 10 minuta.
Reagens v	50	50
Uzorak	200
Temeljito promiješajte, centrifuga na 4000 × g za 10 minutes at room temperature, uzmi supernatanta.

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

Reagens (μL)	Prazna cijev (B)	Standard (S)	Kontrola (C)	Epruveta (T)
0.5 μmol/mL standard phosphorus liquid		100
Supernatant			100	100
Destilirana voda	100
Reagents for determining phosphorus content	1000	1000	1000	1000

Temeljito promiješajte, then place the mix solution in a 40℃water bath for 10 minuta. Cooling to room temperature and detect the absorbance at 660 NM. The blank tube and standard tube just need one or two tubes.

Iii. Izračunavanje:

1. Serum:

Definicija jedinice: 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. Tkivo, bacteria, ili stanice

• Koncentracija proteina:

Definicija jedinice: 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

• Težina uzorka:

Definicija jedinice: 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 težina)=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

• Bakterije ili stanice

Definicija jedinice: 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 stanice ili bakterije.

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;

Vrpca: Ukupni volumen reakcije, 0.5 ml; Vs: Volumen uzorka, 0.2 ml;

CPR: Koncentracija proteina uzorka (mg/ml); T: Vrijeme reakcije (min), 1/6 sat;

W: Težina uzorka (g);

Vl: Volume of reagent I, 1 ml;

500: The amount of bacteria or cell, 5 milijun.

Bilješka

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.

Eksperimentalni primjer:

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 masa) = = 7.5 × ΔAT ÷ΔAS ÷ W = 100.32 U/g masa.

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 masa) = 7.5×ΔAT ÷ ΔAS ÷ W = 2.47 U/g masa.

Reference

[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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