Solarbio β-galactosidase (β-GAL) Komplet za analizu

β-galactosidase (β-GAL) Komplet za analizu

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

Operativna oprema: Microplate Reader/Spectrophotometer

Catalog Number: BC2585

Veličina:100T/48s

Komponente:

Otopina ekstrakta: Liquid 100 ml × 1. Skladištenje na 4 ℃.

Solution I: Prah × 1. Skladištenje na -20 ℃. Dodati 2.55 mL of distilled water to per bottle before use and dissolve it fully. The left reagent store at -20℃.

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

Solution Ⅲ: Liquid 15 ml × 1. Skladištenje na 4 ℃.

Standard: Liquid 1 mL ×1. Skladištenje na 4 ℃. 5 μmol/mL p-nitrophenol solution.

Opis proizvoda

β-galactosidase (β-GAL, EC 3.2.1.23) is an enzyme found broadly in animals, biljke, mikroorganizmi i kultivirane stanice, 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. U ovom kompletu, the β-GAL activity is quantified by measuring the increase in the color development at 400 NM.

Potrebni reagensi i oprema, ali nisu pruženi.

Microplate reader or spectrophotometer, centrifuga, water-bath, PRIJENOS, micro glass cuvette/96 well flat-bottom plate, led, mortar/homogenizer and distilled water.

Postupak

Ja. Preparation of standard samples:

1. Bakterije ili stanice

Collecting bacteria or cells into the centrifuge tube, after centrifugation discard supernatant. Suggest add 1 ml otopine ekstrakta u 5 million of bacteria or cells. Use ultrasonication to splitting bacteria and cells (stavljen na led, ultrasonic power 20%, working time 3 sekundi, interval 10 sekundi, ponoviti za 30 vrijeme). Centrifuge at 15000×g for 20 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

2. Tkivo

Dodati 1 ml otopine ekstrakta u 0.1 g tkiva, and fully homogenized on ice bath. Centrifuge at 15000×g for 20 minuta u 4 ℃ za uklanjanje netopljivih materijala, i prije testiranja uzmite supernatant na ledu.

Ii. Odlučnost

1. Preheat the microplate reader or spectrophotometer for more than 30 minuta, Podesite valnu duljinu na 400 NM, Postavite nulu s destiliranom vodom.
2. Standard Dilute the solution to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/mL with distilled water.

3. Dodajte reagense sa sljedećim popisom:

Temeljito promiješajte. 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. Izračunavanje

• Tissue protein concentration

Definicija jedinice: 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, svaki Mg protein.

β-GAL Activity(U/Mg Prot)= =(y×Vrv)÷(VS × CPR)÷T=14×y÷Cpr

• Tissue weight

Definicija jedinice: 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, svaki uzorak.

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

• Bacteria or cultured cells

Definicija jedinice: 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, svaki 104 Bakterije ili stanice.

β-GAL Activity(U/104 ćelija)= =(y×Vrv)÷(500×Vs÷Ve)÷T=0.028×y

CPR: Supernatant sample protein concentration (mg/ml); Vrpca: Ukupni volumen reakcije, 0.07 ml;

Vs: Supernate volume, 0.01 ml; VE: Extract solution volume,1 ml;

T: Vrijeme reakcije (min), 30 minutes = 0.5 sat; W: Težina uzorka, g;

500: 5 Milijun stanica ili bakterije.

Povezani proizvodi：

BC0340/BC0345 Glucogen Content Assay Kit

BC0360/BC0365 β-1,3-glucanase(β-1,3-GA) Kit za ispitivanje aktivnosti