Solarbio β-galactosidase (β-GAL) Assay-Kit

β-galactosidase (β-GAL) Assay-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Betriebsausrüstung: Microplate Reader/Spectrophotometer

Katalognummer: BC2585

Größe:100T/48S

Komponenten:

Lösung extrahieren: Flüssig 100 ml×1. Lagerung bei 4℃.

Solution I: Pulver×1. Lagerung bei -20℃. Hinzufügen 2.55 mL of distilled water to per bottle before use and dissolve it fully. The left reagent store at -20℃.

Solution Ⅱ: Flüssig 4 ml×1. Lagerung bei 4℃.

Solution Ⅲ: Flüssig 15 ml×1. Lagerung bei 4℃.

Standard: Flüssig 1 mL ×1. Lagerung bei 4℃. 5 μmol/mL p-nitrophenol solution.

Produktbeschreibung

β-galactosidase (β-GAL, EC 3.2.1.23) is an enzyme found broadly in animals, Pflanzen, Mikroorganismen und kultivierte Zellen, which can catalyze the hydrolysis of β-galactosyl bonds and also has the function of transglycosylation. β-GAL can release stored energy for the rapid growth of plants, also catalyzes the degradation of polysaccharides, glycoproteins, and galactose terminal galactose residues in normal polysaccharide metabolism, cell wall component metabolism and during aging cell wall to release free galactose.

β-GAL can catalyze the p-nitro phenyl-β-pyran galactoside to p-nitro phenol. The product has characteristic of absorption at 400 nm. In this kit, the β-GAL activity is quantified by measuring the increase in the color development at 400 nm.

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung.

Microplate reader or spectrophotometer, Zentrifuge, Wasserbad, Pipette übertragen, Mikroglasküvette/96-Well-Platte mit flachem Boden, Eis, mortar/homogenizer and distilled water.

Verfahren

ICH. Preparation of standard samples:

1. Bakterien oder Zellen

Sammeln von Bakterien oder Zellen in die Zentrifugenröhre, after centrifugation discard supernatant. Suggest add 1 mL of Extract solution to 5 million of bacteria or cells. Use ultrasonication to splitting bacteria and cells (auf Eis gelegt, ultrasonic power 20%, working time 3 Sekunden, Intervall 10 Sekunden, Wiederholen Sie 30 mal). Centrifuge at 15000×g for 20 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

2. Gewebe

Hinzufügen 1 mL of Extract solution to 0.1 G Gewebe, and fully homogenized on ice bath. Centrifuge at 15000×g for 20 Minuten bei 4 ℃, um unlösliche Materialien zu entfernen, und nehmen Sie den Überstand vor dem Testen auf Eis.

II. Bestimmung

1. Preheat the microplate reader or spectrophotometer for more than 30 Protokoll, Passen Sie die Wellenlänge an 400 nm, Mit destilliertem Wasser auf Null stellen.
2. Standard Dilute the solution to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/mL with distilled water.

3. Fügen Sie Reagenzien mit der folgenden Liste hinzu:

Gründlich mischen. Detect the absorbance of each tube at 400 nm and noted as AT, AC, AS and AB. Calculate

ΔAT= AT – AC, ΔAS = AS – AB. Each test tube should be provided with one contrast tube.

III. Calculate:

1. Standard curve

Standard curve established: According to the concentration of the standard tube (y nmol/mL) and absorbance ΔAS= AS – AB (X), establish the standard curve. Add ΔA into the standard curve, and calculate the amount of product generated by the sample (Nmol/ml).

2. Berechnung

• Tissue protein concentration

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 nmol of p-nitrophenol in the reaction system per hour, every mg protein.

β-GAL Activity(U/mg-Schutz)=(y×Vrv)÷(Vs×Cpr)÷T=14×y÷Cpr

• Tissue weight

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 nmol of p-nitrophenol in the reaction system per hour, every g sample.

β-GAL Activity(U/g )= (y×Vrv)÷(W×Vs÷Ve)÷T=14×y÷W

• Bacteria or cultured cells

Einheitendefinition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 nmol of p-nitrophenol in the reaction system per hour, every 104 bacteria or cells.

β-GAL Activity(U/104 -Zelle)=(y×Vrv)÷(500× vs ÷ ve)÷T=0.028×y

Cpr: Supernatant sample protein concentration (mg/ml); Seil: Gesamtreaktionsvolumen, 0.07 ml;

Vs: Über den Überblick, 0.01 ml; Ve: Lösungsvolumen extrahieren,1 ml;

T: Reaktionszeit (Mindest), 30 minutes = 0.5 Stunde; W: Probengewicht, G;

500: 5 Millionen Zellen oder Bakterien.

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