Solarbio Laktat Dehidrogenase (LDH) Kit Uji Aktivitas

Dehidrogenase laktat (LDH) Kit Uji Aktivitas

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

Peralatan Operasi: Spektrofotometer/pembaca lempeng mikro

Kucing No: BC0685

Ukuran:100T/48S

Komponen:

Extract solution: 60 mL×1. Penyimpanan pada suhu 4℃；

Reagen I: 7 mL×1. Penyimpanan pada suhu 4℃.

Reagen II: powder×1. Storage at -20℃. Solusi kerja: dissolve with 1.3 mL air suling sebelum digunakan. It can be divided into tubule after matching, which can be preserved for two weeks at -20℃. Avoid repeated freeze and thaw cycles.

Reagen III: 7 mL×1. Penyimpanan pada suhu 4℃.

Reagen IV: 25 mL×1. Penyimpanan pada suhu 4℃.

Sodium pyruvate standard solution: 1 ml (2 μmol/mL) ×1. Penyimpanan pada suhu 4℃.

Deskripsi Produk:

Lactate dehydrogenase (LDH or LD) is the terminal enzyme of the glycolysis pathway which is found in nearly all living cells (animals, plants, and prokaryotes). LDH catalyzes the conversion of lactate to pyruvic acid and back, as it converts NAD+ to NADH and back.

NAD+ and lactic acid are oxidized to pyruvic acid by the catalysis of LDH. Pyruvate further reacted with 2,4-dinitrophenylhydrazide to form pyruvate dinitrobenzone, which show brown red color in alkaline solution and the color depth is proportional to the concentration of pyruvate.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:

Spectrophotometer/Microplate reader, thermostat water bath, mesin sentrifugal meja, pipet yang dapat disesuaikan, kuvet kaca mikro/pelat dasar datar sumur 96, mortir/homogenizer, Es, air suling.

Prosedur:

SAYA. Sampel preparation:

1. Bacteria, cells, or tissue sample:

Bakteri atau sel:

Collecting bacteria or cells into the centrifuge tube. The liquid in the upper layer is discarded after centrifugation. The ratio of bacteria/cell amount (104): Extract solution volume (ml) is 500~1000:1(it is suggested to take about 5 million bacteria/cell and add 1 mL of Extract solution). Bacteria and cell is split by ultrasonic (ditempatkan di atas es, 200W, work time 3s, interval 10s, ulangi untuk 30 waktu). Sentrifugasi di 8000 rpm 4℃ for 10 menit, take the supernatant and put it on ice for test.

Jaringan:

Ice-bath homogeneity is conducted according to the ratio of tissue mass (G): Extract solution volume (ml) = 1: 5~10 (it is recommended to take about 0.1 g of tissue and add 1 mL of Extract solution). Ice bath homogenization. Sentrifugasi di 8000 rpm and 4℃ for 10 menit, take the supernatant and put it on ice for test.

2. Serum (plasma)sample:

Detect sample directly.

II. Prosedur:

1. Preheat the Spectrophotometer/Microplate reader 30 menit, sesuaikan panjang gelombang ke 450 nm, atur nol dengan air suling.
2. Sodium pyruvate standard solution: mengambil 100 μL of standard solution, dilute to 1, 5, 0.25, 0.125, 0 μmol/mL, and use 2, 1, 0.5, 0.25, 0.125, 0 μmol/mL as standard curve.

3. Sample Test

Mixed thoroughly, place at room temperature for 3 menit. Mengambil 200 μL of reaction solution in micro glass cuvette/96 well flat-bottom plate, measured the absorbance at 450 nm, ΔA=AT-AC. Each test tube should set a control tube.

AKU AKU AKU. LDH Perhitungan.

1. Sample Sodium pyruvate content
2. Set the standard curve, y-axis as the standard concentration, μmol/mL, x-axis as the 450 nm absorption. Put ΔA(X) into standard curve, calculate y(μmol/mL)
3. Serum (plasma) sample LDH activity

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, every milliliter of serum.

LDH(U/mL)=y÷T×103=66.7×y

4. Jaringan, bacteria or cultured cells LDH activity

A. Konsentrasi protein:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, every milligram of protein.

LDH(U/mg keuntungan)= y÷T÷Cpr×103 =66.7×y÷Cpr

B. Berat sampel

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, in every gram of tissue. LDH(U/g)= y÷T÷W×103 =666.7×y

C. Cell amount

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute every 10000 cells.

LDH(U/104 cell)= y÷T÷500×103 =0.133×y

Vs: Supernate volume (ml), 0.01 ml; Vsv: Extract solution volume, 1 ml; T: Waktu reaksi, 15 menit;

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

500: Total number of bacteria or cells, 5 juta; 103: 1 μmol/mL=103 nmol/mL.

Referensi

[1] Huang P H, Fu L C, Huang C S, dkk. The uptake of oligogalacturonide and its effect on growth inhibition, lactate dehydrogenase activity and galactin-3 release of human cancer cells[J]. Food chemistry, 2012, 132(4): 1987-1995.

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