Kit di analisi dell'attività Ca++Mg++-ATPasi Solarbio

Ca++Mg++-ATPase Activity Assay Kit

Nota: Prendi due o tre campioni diversi per la previsione prima del test.

Attrezzatura operativa: Spettrofotometro

Gatto n: BC0960

Misurare: 50T/24S

Componenti:

Reagente I: Liquido 30 ml×1. Conservazione a 4 ℃.

Reagente II: Liquido 4 ml×1. Conservazione a 4 ℃.

Reagente III: Polvere×2. Conservazione a -20 ℃. Dissolve thoroughly with 1 ml di acqua distillata prima dell'uso. The rest reagent can be kept at -20℃ for one week.

Reagente IV: Liquido 2 ml×1. Conservazione a 4 ℃.

Reagente V: Polvere×1. Conservazione a 4 ℃. Dissolve thoroughly with 3 ml di acqua distillata prima dell'uso. Reagente VI: Polvere×1. Conservazione a 4 ℃. Dissolve thoroughly with 15 ml di acqua distillata prima dell'uso, can be kept at 4℃ for one week.

Reagent VII: Polvere×1. Conservazione a 4 ℃. Dissolve thoroughly with 15 ml di acqua distillata prima dell'uso, can be kept at 4℃ for one week.

Reagent VIII: Liquido 15 ml×1. Storage at RT.

Soluzione standard: Liquido 1 ml×1. 10 μmol/mL standard phosphorus liquid, Archiviazione a 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. Per esempio: aggiungere 1.9 ml di acqua distillata a 0.1 mL of standard, mescolare accuratamente.

Phosphorus fixing reagent:

Prepare reagents for determining phosphorus content: make solution as the volume ratio of H2O: Reagente 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.

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

Descrizione del prodotto:

Ca++Mg++-ATPase is widely distributed in plants, animali, 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.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, centrifuga da tavolo, pipetta regolabile, bagnomaria, 1 Cuvetta di vetro da ml, mortaio/omogeneizzatore, ghiaccio e acqua distillata.

 

Procedura:

IO. preparazione del campione:

1. Batteri o cellule:

Collecting bacteria or cells into a centrifuge tube, centrifugation, and discard supernatant. Suggest add 1mL of Reagent I to 5 milioni di batteri o cellule. Use ultrasonic to splitting bacteria and cells (posto sul ghiaccio, ultrasonic power 20%, working time 3 secondi, intervallo 10 secondi, ripetere per 30 volte). Centrifugare a 8000 ×g per 10 minutes at 4℃ and take the supernatant on ice before testing.

2. Tessuto:

Aggiungere 1 mL of Reagent I into 0.1 g di tessuto, macinazione completa su ghiaccio. Centrifugare a 8000 ×g per 10 minutes at 4℃ and take the supernatant on ice before testing.

3. Siero: Directly

II. Determinazione:

1. Preriscaldare lo spettrofotometro per 30 minuti, regolare la lunghezza d'onda su 660 nm, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:

Reagente (μL)	Tubo di controllo (C)	Provetta (T)
Reagente I	130	90
Reagente II	80	80
Reagente III	40	40
Reagente IV		40
Campione		200
Mescolare accuratamente, then place the reaction solution in a 37℃ (mammifero) o 25 ℃ (altre specie) bagnomaria per 10 minuti.
Reagente V	50	50
Campione	200
Mescolare accuratamente, centrifugare a 4000 ×g per 10 minutes at room temperature, prendi il surnatante.

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

Reagente (μL)	Tubo vuoto (B)	Tubo standard (S)	Tubo di controllo (C)	Provetta (T)
0.5 μmol/mL standard phosphorus liquid		100
Supernatante			100	100
Acqua distillata	100
Reagents for determining phosphorus content	1000	1000	1000	1000

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

III. Calcolo:

1. Siero:

Definizione di unità: 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. Tessuto, batteri, O cellule

• Concentrazione proteica:

Definizione di unità: 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

• Peso del campione:

Definizione di unità: 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 (Peso 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

• batteri o cellule

Definizione di unità: 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 (Cella U/104 )=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;

Corda: Volume totale di reazione, 0.5 ml; Contro: Volume del campione, 0.2 ml;

Cpr: Concentrazione proteica del campione (mg/ml); T: Tempo di reazione (min), 1/6 ora;

W: Peso del campione (G);

Vl: Volume of reagent I, 1 ml;

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

Nota

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.

Esempio sperimentale:

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

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

Riferimenti

[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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