Solarbio Pyrophosphate: Fructose 6-phosphate-1 Phosphotransferase Assay Kit

Komponen:

Ekstrak larutan: 55mL × 1, stored at 4 °C.

Reagen 1: 40mL × 1, stored at 4 °C and protected from light.

Reagen 2: Bubuk × 1, stored at -20 °C and protected from light. Just before use, menambahkan 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C untuk 1 week after dispensing to avoid repeated freeze-thaw cycles.

Reagen 3: powder × 1, stored at -20 °C and protected from light. Just before use, menambahkan 6 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C after dispensing. Prohibition of repeated freeze-thaw cycles.

Reagen 4: 91µL × 2, stored at 4 °C and protected from light. Just before use, menambahkan 0.209 mL of distilled water to fully dissolve. Stored at 4 °C untuk 1 pekan.

Reagen 5: Bubuk × 2, stored at -20 °C and protected from light. Just before use, menambahkan 0.3 mL of distilled water to fully dissolve. Unused reagents are stored at -20 °C untuk 1 week after dispensing.

Reagen 6: 60 µL × 1, stored at 4 °C and protected from light. Just before use, menambahkan 0.6 mL of distilled water to fully dissolve. Stored at 4 °C untuk 1 pekan.

Deskripsi Produk:

Pyrophosphate: Fructose-6-phosphate-1-phosphotransferase (PFP, EC2.7.1.90) is a cytosolic enzyme that is widely present in plant tissues and catalyzes the phosphorylation of fructose-6-phosphate like phosphofructokinase. As a result, the single PEP catalytic reaction is a reversible reaction, and pyrophosphate is used instead of ATP, which plays an important role in carbon metabolism of photosynthesis.

PFP catalyzes the conversion of fructose 6-phosphate to fructose 1,6-diphosphate, which is converted to dihydroxyacetone phosphate by the action of aldolase and triose phosphate isomerase, and then catalyzed by α-phosphate glycerol dehydrogenase and NADH to from Glycerol 3-diphosphate and NAD. The change in absorbance at 340 nm reflects the level of PFP activity.

Required material

Low temperature centrifuge, spektrofotometer, water bath/constant temperature incubator, mortir/homogenizer, 1 mL kuvet kuarsa, pipet transfer, es dan air suling, tabung EP.

Prosedur:

SAYA. Sampel Extraction:

1. 1. Sampel jaringan:

According to the mass of the tissue (G): the volume of the extract solution (ml) adalah 1: 5 ~ 10. Suggested 0.1g of tissue with 1mL of extract solution. Fully grind on ice, centrifugated at 20000g and 4℃ for 15 menit. Supernatant is placed on ice for test.

2. Bakteri atau sel:

According to the number of cells (104): the volume of the extract solution (ml) adalah 500 ~ 1000: 1. Recommend 5 million with 1mL of Extract Solution. Use ultrasonication to split bacteria or cells (power 300W, Waktu kerja 3s, interval 7 detik, waktu keseluruhan 3 menit). Centrifugated at, 20000g and 4℃ for 15 menit. Supernatant is placed on ice for test.

3. Liquids: direct detection.

II. Tekad prosedur:

• Memanaskan lebih dulu spektrofotometer 30 menit, sesuaikan panjang gelombang ke 340 nm, atur nol dengan sulingan
• Tambahkan reagen dengan daftar berikut:

AKU AKU AKU. Calculation of PFP activity:

1 Calculated by micro quartz cuvette

• Calculated by protein concentration:

Definisi satuan: One unit of enzyme activity is defined as that 1 mg of tissue protein per minute consumes 1 nmol of NADH.

PFP activity (U/mg keuntungan) =ΔA÷（ε×d）×VRT×109÷(VS×Cpr) ÷T=53.59×ΔA÷Cpr

• Calculated by sample weight

Definisi satuan: One unit of enzyme activity is defined as that 1g of tissue per minute consumes 1 nmol of NADH.

PFP activity (U/g berat segar) =ΔA÷（ε×d）×VRT×109÷(W ×VS÷VE) ÷T=53.59×ΔA÷W

• Calculated by bacteria or cell amount:

Definisi satuan: One unit of enzyme activity is defined as that 10 thousand bacteria or cells per minute consumes 1 nmol of NADH.

PFP activity (sel U/104) =ΔA÷（ε×d）×VRT×109÷(VS×N÷VE) ÷T=53.59×ΔA÷N（104）

• Calculated by serum and other liquids:

Definisi satuan: One unit of enzyme activity is defined as that 1 mL of liquids per minute consumes 1 nmol of NADH.

PFP activity (U/mL) =ΔA÷（ε×d）×VRT×109÷VS÷T=53.59×ΔA

Vrt: total volume of reaction system, 0.001 L;

e: Koefisien kepunahan molar NADH, 6.22 × 103 L/mol/cm; D: cuvette light path, 1 cm;

Vs.: added sample volume, 0.1 ml;

VE: volume of extract solution added, 1 ml; T: waktu reaksi, 30 menit;

Cpr: Konsentrasi protein sampel, mg/mL; W: massa sampel, 0.1 G;

109:conversion factor, 1 mol = 109 nmol N: number of cell

2. Calculated by 96-well UV plate:

Modify d = 1 cm in the above formula to d-0.6 cm (the light path of a 96-well plate) for calculation.

Catatan:

1. The number of samples should not be too large to avoid delaying the enzymatic reaction time.

Contoh eksperimental:

1. Mengambil 0.1 g of bean sprouts and add 1 mL of Extract solution for sample processing. After centrifugation to take the supernatant, proceed according to the determination procedure. CalculateΔA = (A1T-A2T)-(A1B-A2B)= (1.041-0.963)-0=0.078. The enzyme activity is calculated according to the sample mass.

PFP activity (U/g berat segar) =53.59×ΔA÷W=41.8 U/g fresh weight.

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