Solarbio Superossido Dismutasi (ZOLLA ERBOSA) Kit di analisi dell'attività

Superoxide Dismutase (ZOLLA ERBOSA) Kit di analisi dell'attività

Nota: It is necessary to predict 2-3 large difference samples before the formal determination.

Attrezzatura operativa: Spettrofotometro

Gatto n: BC0170

Misurare: 50T/24S

Componenti:

Extraction reagent: 60 ml×1. Conservazione a 4 ℃.

Reagent Ⅰ: 15 ml×1. Conservazione a 4 ℃.

Reagent Ⅱ: 160 μL×1. Conservazione a 4 ℃. Mix by pipetting after centrifugation.

Reagent Ⅲ: 11 ml×1. Conservazione a 4 ℃.

Reagent Ⅳ: Polvere×1. Conservazione a 4 ℃.

Reagente V: 2 ml×1. Conservazione a 4 ℃. Add Reagent Ⅳ to Reagent V before use and shake with an oscillator to mix thoroughly. It can be stored 3 mesi.

Descrizione del prodotto:

Superoxide dismutase (ZOLLA ERBOSA, CE 1.15.1.1) è ampiamente presente negli animali, impianti, microrganismi e cellule in coltura. It catalyzes the superoxide anion to form H2O2 and O2. SOD is not only the superoxide anion scavenging enzyme, but also the main H2O2 producing enzyme, which plays an important role in the biological antioxidant system.

Superoxide anion (O2-) is produced by the xanthine and xanthine oxidase reaction system. O2- can reduce blue tetrazole to create blue formazan, which has absorbance in 560 nm. SOD can remove O2- and inhibit the formation of methionine. The darker the blue color of the reaction solution, the lower the activity of SOD. The lighter the blue color of the reaction solution, the higher the activity of SOD.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, centrifuga da tavolo, transfer pipette, 1 Cuvetta di vetro da ml, mortaio/omogeneizzatore, ghiaccio e acqua distillata.

Operation steps:

IO. preparazione del campione:

1. Batteri o cellule: collecting bacteria or cells into the centrifuge tube, discard supernatant after centrifugation. According to the proportion of bacteria or cells (104 cellule): extraction solution volume (ml) Di 1:5-10 to extract. It is suggested that 5 million of bacteria or cells amount with 1mL of Extraction reagent. Splitting the bacteria or cells with ultrasonication (placed on ice, ultrasonic power 200W or 20%, working time 3s, intervallo 10s, ripetere per 30 volte). Centrifugare a 8000 ×g per 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
2. Tessuto: according to the proportion of tissue weight (G): extraction solution volume (ml) Di 1:5-10 to extract. It is suggested that 0.1 g di tessuto con 1 mL of Extraction reagent and fully homogenized on ice.
3. Centrifugare a, 8000 ×g per 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
4. Siero (plasma) campione: detect sample directly.

II. Determinazione procedura:

1. Preriscaldare lo spettrofotometro per 30 minuti, regolare la lunghezza d'onda su 560 nm e azzerare con acqua distillata.
2. Keep Reagent Ⅰ, Reagent Ⅲ, Reagent V in water bath for more than 5 minuti a 37℃(mammifero) O

25℃ (altre specie).

3. Aggiungere i reagenti con il seguente elenco:

Mix thoroughly and the mixture is incubated at room temperature for 30 minuti. Add the mixture into 1mL glass cuvette, and detect the absorbance value of each tube at 560 nm. ΔAT=AT-AC，ΔAB=AB1-AB2. If there is precipitation at the bottom, mix thoroughly and then measure.

III. Calcolo:

1. Inhibition percentage:

Inhibition percentage=[ΔAB -ΔAT]÷ΔAB× 100%

The inhibition percentage should be in 30%~70% (the value close to 50% will have a more accurate result). If the calculated inhibition percentage is less than 30% o più di 70%, it is usually necessary to adjust the sample addition amount and re determine. If the percentage of inhibition is too high, il campione deve essere diluito adeguatamente. If the percentage of inhibition is too low, the sample should be reprepared with a higher concentration.

2. Definizione di unità: One unit of enzyme activity is defined as the amount of enzyme catalyzes the inhibition of 50% in the reaction system of the above xanthine
3. Calcolo

UN. Siero (plasma)campione

ZOLLA ERBOSA (U/ml)=[P÷(1-P)×Vrv]÷Vs×F=11.4×P÷(1-P)×F

B. Tessuto, bacteria or cultured cells

UN) Concentrazione proteica:

ZOLLA ERBOSA (U/mL prot) = [P÷(1-P)×Vrv]÷(Vs×Cpr)×F=11.4×P÷(1-P)÷Cpr×F

B) Peso del campione

ZOLLA ERBOSA (Peso U/g) = [P÷(1-P)×Vrv]÷(W×Vs÷Vsv)×F=11.4×P÷(1-P)÷W×F

c) Bacteria or cell amount

ZOLLA ERBOSA (Cella U/104)=[P÷(1-P)×Vrv]÷(500×Vs÷Vsv)×F=0.0228×P÷(1-P)×F

Corda: Volume totale di reazione, 1.026 ml; Contro: Volume del campione, 0.09 ml;

Vsv: Extraction volume, 1 ml;

Cpr: Concentrazione proteica del campione, mg/ml; W: Peso del campione, G;

500: Total number of bacteria and cells, 5 milioni. P: Inhibition percentage, %;

F: Sample dilution multiple.

Nota:

1. The Sample and Reagent Ⅱ should be placed on ice when
2. When there are many samples, the working solution (including Reagent I, II and III) can be configured according to the table. Reagent V must be added
3. After the reaction completed, there may be precipitation formed, which can be determined after

Esempi sperimentali:

1. 1 g of Echinochloa crusgalli is added into 1 mL of Extraction reagent for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT = AT – AC = 0.335-0.012 = 0.323, ΔAB = AB1 – AB2 = 0.957-0.003 = 0.954. Inhibition percentage = (ΔAB- ΔAT)÷ ΔAB × 100% = 72%, and the enzyme activity is calculated according to the sample mass.

SOD activity (Massa U/g) = 11.4 × Inhibition percentage (1-Inhibition percentage) × W = 293.14 Massa U/g.

2. 1 mL of Extraction reagent is added to 0.1 g of rat spleen for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT= AT- AC = 0.563-0.213 = 0.35, ΔAB= AB1- AB2= 0.957-0.003 = 0.954, inhibition percentage = (ΔAB -ΔAT)÷ ΔAB×100% =31%

SOD activity (Massa U/g) = 11.4 × Inhibition percentage (1-Inhibition percentage) ×W = 196.71 Massa U/g.

3. 10 million cells is extracted and centrifuged by adding 1 mL of Extraction reagent, and then the operation is performed according to the determination steps. The results are as follows: ΔAT =AT – AC=614-0.015 = 0.599, ΔAB= AB1- AB2= 0.944-0.005 = 0.939, inhibition percentage = (ΔAB- ΔAT) ×ΔAB× 100% = 36.21%

SOD activity (Cella U/104) = Inhibition percentage ÷ (1-Inhibition percentage)× VTS]÷(1000× VS ÷ VTS) = 0.0065 Cella U/104.

Riferimenti:

• Spitz D R, Oberley L W. An assay for superoxide dismutase activity in mammalian tissue homogenates[J]. Biochimica analitica, 1989,179(1):8-18.
• Masayasu M, Hiroshi Y. A simplified assay method of superoxide dismutase activity for clinical use[J]. Clinica Chimica Acta, 1979,92(3):337-342.

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