Solarbio β-galactosidase (β-GAL) Perangkat Pengujian

β-galactosidase (β-GAL) Perangkat Pengujian

Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.

Peralatan Operasi: Microplate Reader/Spectrophotometer

Nomor Katalog: BC2585

Ukuran:100T/48S

Komponen:

Ekstrak larutan: Cairan 100 mL×1. Penyimpanan pada suhu 4℃.

Solution I: Bedak×1. Penyimpanan pada -20℃. Menambahkan 2.55 mL of distilled water to per bottle before use and dissolve it fully. The left reagent store at -20℃.

Solution Ⅱ: Cairan 4 mL×1. Penyimpanan pada suhu 4℃.

Solution Ⅲ: Cairan 15 mL×1. Penyimpanan pada suhu 4℃.

Standar: Cairan 1 Ml × 1. Penyimpanan pada suhu 4℃. 5 μmol/mL p-nitrophenol solution.

Deskripsi Produk

β-galactosidase (β-GAL, EC 3.2.1.23) is an enzyme found broadly in animals, tanaman, mikroorganisme dan sel yang dikultur, 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. Dalam paket ini, the β-GAL activity is quantified by measuring the increase in the color development at 400 nm.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan.

Pembaca pelat mikro atau spektrofotometer, alat sentrifugal, pemandian air, pipet transfer, kuvet kaca mikro/pelat dasar datar sumur 96, Es, mortar/homogenizer and distilled water.

Prosedur

SAYA. Preparation of standard samples:

1. Bakteri atau sel

Mengumpulkan bakteri atau sel ke dalam tabung centrifuge, after centrifugation discard supernatant. Suggest add 1 mL larutan Ekstrak ke 5 juta bakteri atau sel. Use ultrasonication to splitting bacteria and cells (ditempatkan di atas es, kekuatan ultrasonik 20%, waktu kerja 3 detik, selang 10 detik, ulangi untuk 30 waktu). Centrifuge at 15000×g for 20 minutes at 4℃ to remove insoluble materials and take the supernatant on ice before testing.

2. Jaringan

Menambahkan 1 mL larutan Ekstrak ke 0.1 g tisu, and fully homogenized on ice bath. Centrifuge at 15000×g for 20 menit pada suhu 4℃ untuk menghilangkan bahan yang tidak larut, dan mengambil supernatan di atas es sebelum pengujian.

II. Tekad

1. Preheat the microplate reader or spectrophotometer for more than 30 menit, sesuaikan panjang gelombangnya 400 nm, atur nol dengan air suling.
2. Standard Dilute the solution to 200, 100, 50, 25, 12.5, 6.25, 0 nmol/mL with distilled water.

3. Tambahkan reagen dengan daftar berikut:

Aduk rata. 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.

AKU AKU AKU. 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. Perhitungan

• Tissue protein concentration

Definisi satuan: 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 keuntungan)=(y×Vrv)−(Vs×Cpr)÷T=14×y÷Cpr

• Tissue weight

Definisi satuan: 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

Definisi satuan: 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, setiap 104 bakteri atau sel.

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

Cpr: Supernatant sample protein concentration (mg/mL); Tali: Volume reaksi total, 0.07 ml;

Vs: Supernate volume, 0.01 ml; Ve: Ekstrak volume solusi,1 ml;

T: Waktu reaksi (menit), 30 minutes = 0.5 jam; W: Berat sampel, G;

500: 5 juta sel atau bakteri.

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