Solarbio Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Kit Uji Aktivitas

Glyceraldehyde-3-phosphate Dehydrogenase(GAPDH) Kit Uji Aktivitas

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

Detection instrument: Spektrofotometer

Kucing No: BC2210

Ukuran: 25T/24 ; 50T/48S

Komponen:

Ekstrak larutan: 60 mL×1. Simpan pada suhu 4℃.

Reagen I: Bedak×1. Simpan pada suhu -20℃.

Reagen II: 50 mL×1. Simpan pada suhu 4℃.

Reagen III: 30 μL×1. Simpan pada suhu 4℃. The liquid is placed in the EP tube in the reagent bottle. According to the dosage and the volume ratio of Reagent III: distilled water of 3:100, campur dengan baik, use and prepare now.

Deskripsi Produk:

GAPDH (EC 1.2.1.12) catalyzes the oxidation of glyceraldehyde 3-phosphate to 1,3-diphosphoglyceride. It is the key enzyme of glycolysis pathway. It is closely related to the pathway of gluconeogenesis, the maintenance of blood glucose concentration and the occurrence of diabetes. It plays an important role in the disorders of glucose, lipid and protein metabolism.

3-phosphoglycerate kinase can catalyze the production of 1,3-diphosphoglyceride from triphosphate and ATP. GAPDH reversely catalyzes the formation of glyceraldehyde-3-phosphate, inorganic phosphorus and NAD+ from 1,3-diphosphoglyceride and NADH. The decrease of NADH measured at 340 nm can reflect the activity of GAPDH.

Required material

Desk centrifuge, ultraviolet spectrophotometer, Bawah Air Suhu Konstan, mortar/ homogenizer, 1 mL kuvet kuarsa, TransferPettor, es dan air suling.

Prosedur:

SAYA. Sampel Extraction:

1. Sampel jaringan:

According to the mass of the tissue (G): volume solusi ekstrak (ml) adalah 1: 5~10. Suggested 0.1 g jaringan dengan 1 ML larutan ekstrak. Fully grind on ice, sentrifugasi di 8000 g and 4℃ for 20 menit. Supernatant is placed on ice for test.

2. Bakteri atau sel:

According to the number of cells (104): volume solusi ekstrak (ml) adalah 500 ~ 1000: 1. Recommend 5 million with 1 mL Larutan Ekstrak. Use ultrasonication to split bacteria or cells (kekuatan 20% or 200W, Waktu kerja 3s, interval 10 detik, ulangi untuk 30 waktu). Sentrifugasi di, 8000 g and 4℃ for 10 menit. Supernatant is placed on ice for test.

3. Serum (plasma): direct measurement.

II. Tekad prosedur:

• Preheat the ultraviolet spectrophotometer 30 menit, adjust wavelength to340 nm, atur nol dengan air suling.
• Preparation of working solution: pour all Reagent II into one bottle of Reagent I. Fully dissolved. Preheat a certain amount of 37℃ (mamalia) atau 25℃ (spesies lain) untuk 10 min as required. The unused reagents shall be stored at — 20℃ after sub charging. Hindari pembekuan dan pencairan berulang.
• Tambahkan reagen dengan daftar berikut:

Nama reagen (μL)	Tabung reaksi (T)	Tabung kosong (B)
Sampel	30
Air sulingan		30
Reagen III	20	20
Solusi kerja	950	950
Tambahkan reagen di atas ke dalam 1 ML kuarsa cuvette masing -masing. Aduk rata. Measure the absorbance value A1 at 340 nm for 10s. Quickly put it into a water bath or incubator at 37℃ (mamalia) atau 25℃ (spesies lain) untuk 5 menit. Take out and dry it quickly. Measure the absorbance value A2 for 5min10s. Calculate ΔAT= A1T- A2T, ΔAB= A1B- A2B, ΔA = ΔAT – ΔAB. Blank tube only needs to test 1–2 times.

AKU AKU AKU. Perhitungan:

Calculated by micro quartz cuvette

• Calculated by protein concentration:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every mg protein.

GAPDH activity (U/mg keuntungan) = Δa ÷(×d)× VRV × 109 ÷(VS×Cpr)÷T=1072×ΔA÷Cpr

• Calculated by sample weight

Definisi satuan: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every g sample.

GAPDH activity (U/g berat segar) = Δa ÷(×d)× VRV × 109 ÷(VS×W÷VST)÷T= 1072×ΔA÷W

• Calculated by bacteria or cell amount:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, setiap 104 bakteri atau sel.

GAPDH activity (sel U/104) = Δa ÷(×d)× VRV × 109 ÷(VS×500÷VST)÷T = 2.14×ΔA

• Calculated by volume of culture medium:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzymes consumes of 1 nmol of NADH in the reaction system per minute, every mL liquid.

GAPDH activity (U/mL) = Δa ÷(×d)×VRV×109÷VS÷T= 1072×ΔA

 

e: molar extinction coefficient of NADH: 6.22× 103 l/mol/cm; D: light path of cuvette, 1 cm;

Tali: volume reaksi total, 0.001 L;

Vs.: sample volume in reaction system, 0.03 ml; Vst: volume of extraction solution added, 1 ml; Cpr: Konsentrasi protein sampel, mg/mL;

W, massa sampel, G;

T: waktu reaksi, 5 menit;

109: conversion factor, 1 mol = 109 nmol;

500: Jumlah sel, 5 juta.

Catatan:

1. When A1 is less than 0.8 or ΔA is greater than 0.7, it is recommended to dilute the sample before determination.
2. The blank tube is a test tube for testing the quality of each reagent component. Under normal conditions, the change does not exceed01.

Contoh eksperimental:

1. Take 0.1g of rabbit kidney, menambahkan 1 ML larutan ekstrak, homogenize in ice bath, then centrifuge at 8000g, 4℃ untuk 20 menit, take the supernatant and put it on ice, then operate with micro quartz cuvette according to the determination steps, measure and calculate: ΔAT=A1T-A2T =0.815-0.158=0.657, ΔAB= A1B – A2B = 0.865-0.864=0.001, ΔA = ΔAT – ΔAB = 656.

GAPDH activity (massa U/g) = 1072×ΔA ÷ W = 7032.32 massa U/g.

2. Take 0.1g of clover, menambahkan 1 ML larutan ekstrak, homogenize it in ice bath, then centrifuge it in 8000g and 4℃ for 20 menit, take the supernatant and put it on ice, then operate according to the determination steps, measure and calculate it with micro quartz cuvette, ΔAT = A1T – A2T =1.026-1.017=0.009, ΔAB= A1B – A2B =0.865-0.864=0.001, ΔA = ΔAT – ΔAB=008.

GAPDH activity (massa U/g) = 1072 × ΔA ÷ W =85.76 U/g mass.

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