Solarbio Tissue and Blood Alkaline Phosphatase (AKP/ALP) Kit di analisi dell'attività

Tissue and Blood Alkaline Phosphatase (AKP/ALP) Kit di analisi dell'attività

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

Attrezzatura operativa: Spettrofotometro

Numero di catalogo: BC2140

Misurare: 50T/24S

Componenti:

Reagente di estrazione: Liquido 30 bottiglia ml × 1, Archiviazione a 4 ℃.

Reagente 1: Liquido 10 bottiglia ml × 1, storage at 4℃ and protect from light.

Reagente 2: Liquido 10 bottiglia ml × 1, storage at 4℃ and protect from light.

Reagente 3: Liquido 30 bottiglia ml × 1, storage at 4℃ and protect from light. It cannot be used if it turns to blue-green.

Standard: Liquido 1 bottiglia ml × 1, 10 μmol/mL phenol standard solution, Archiviazione a 4 ℃. Dilute with distilled water to 2.5 μmol/mL before use.

Descrizione del prodotto:

AKP/ALP is a zinc-containing glycoprotease, which hydrolysis various natural and synthetic phospholipid monoester compounds in alkaline condition. AKP / ALP are widely distributed in human organs, mainly in liver.

In alkaline condition, AKP/ALP catalyzes hydrolysis of disodium phenyl phosphate to phenol, and the phenol reacts with 4-Aminoantipyrine and potassium ferricyanide to form red quinone derivative, which can be detected absorbance at 510 nm. AKP/ALP activity can be calculated by measuring the absorbance increase rate at 510 nm.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, centrifuga da tavolo, trasferente, 1 Cuvetta di vetro da ml, ghiaccio e acqua distillata.

Procedura:

IO. Enzima preparazione:

Aggiungere 1 mL Extraction reagent to 0.1 g tissue, grind thoroughly. Centrifuge at 4℃, 10000 giri al minuto per 10 minuti. Take the supernatant on the ice for test. Blood sample can be detected directly. Dilute with Extraction reagent if concentrate is high.

II. Determinazione procedura:

1. Preriscaldare lo spettrofotometro per 30 minuti, regolare la lunghezza d'onda su 510 nm, Imposta il contatore su zero con distillazione
2. Add reagents in 1.5 mL cuvette as the following:

Nome del reagente (μL)	Test tube A3	Control tube A4	Blank tube A2	Standard tube A1
Acqua distillata			20
Soluzione standard				20
Supernatante	20
Reagente 1	200	200	200	200
Reagente 2	200	200	200	200
Mescolare accuratamente, stay in 37℃ for 15 minuti.
Reagente 3	600	600	600	600
Supernatante		20
Mescolare accuratamente, detect absorbance at 510 nm, record as A1, A2, A3, A4.

III. AKP/ALP activity calculation:

• Concentrazione proteica:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every mg protein.

AKP/ALP (U/mg prot) =[C×(A3-A4)÷(A1-A2)×Vs]÷(Cpr×Vs)÷T=0.167×(A3-A4)÷(A1-A2)÷Cpr

• Peso del campione:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every g sample.

AKP/ALP (Peso U/g) =[C×(A3-A4)÷(A1-A2)×Vs]÷(W÷Ve×Vs)÷T=0.167×(A3-A4)÷(A1-A2)÷W

• Sangue:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every mL serum.

AKP/ALP (U/ml) =[C×(A3-A4)÷(A1-A2)×Vs]÷Vs÷T=0.167×(A3-A4)÷(A1-A2)

C: Concentrazione standard, 2.5 µmol/ml;

Corda: Volume totale di reazione, 1020 μL=1.02 mL; Contro: Supernatant volume, 0.02 ml;

Ve: Volume di estrazione, 1 ml; W: Peso del campione, G;

T: Tempo di reazione, 15 minuti;

Cpr: Concentrazione proteica del campione, mg/ml.

Nota:

1. Reagente 1, Reagente 2 and Reagent 3 should be protected from
2. Reagente 3 cannot be used if it has changed toblue-green.
3. Mix thoroughly quickly after adding Reagent 3 to avoid incomplete

Esempio sperimentale:

1. Take 0.1g of mouse pancreas and add 1 mL of Extract solution for homogenate. After taking the supernatant, operate according to the determination steps. Calculate AT = 0.324, AC = 0.037, AB = 0.022, AS = 0.677. Calculate the enzyme activity according to the sample mass: AKP/ALP enzyme activity (Massa U/g) = 0.167×(AT-AC)÷(As-ab) ÷ W =0.167×(0.324-0.037)÷(0.677-0.022)÷0.1=0.732 U/g
2. After taking the rabbit serum, the operation is carried out according to the determination steps, and the enzyme activity is calculated as follows: AT = 0.190, AC = 0.015, AB = 0.022, AS = 0.677. According to the blood volume, l'attività enzimatica viene calcolata come segue: AKP/ALP enzyme activity (U/ml) = 0.167×(AT-AC) ÷ (As-ab) =0.167×(0.190-0.015)÷(0.677-0.022)=0.0446U/mL.

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