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:

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

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

Reagen II: bubuk × 1. Penyimpanan pada -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 mol/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 (binatang, tanaman, 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 persiapan:

1. Bakteri, sel, or tissue sample:

Bakteri atau sel:

Mengumpulkan bakteri atau sel ke dalam tabung centrifuge. The liquid in the upper layer is discarded after centrifugation. The ratio of bacteria/cell amount (104): Ekstrak volume solusi (ml) adalah 500 ~ 1000:1(disarankan untuk mengambil alih 5 juta bakteri/sel dan tambahkan 1 ML larutan ekstrak). Bacteria and cell is split by ultrasonic (ditempatkan di atas es, 200W, Waktu kerja 3s, interval 10 detik, ulangi untuk 30 waktu). Sentrifugasi di 8000 rpm 4℃ for 10 menit, take the supernatant and put it on ice for test.

Jaringan:

Homogenitas pematangan es dilakukan sesuai dengan rasio massa jaringan (G): Ekstrak volume solusi (ml) = 1: 5~10 (it is recommended to take about 0.1 g jaringan dan tambahkan 1 ML larutan ekstrak). Ice bath homogenization. Sentrifugasi di 8000 rpm dan 4 ℃ untuk 10 menit, take the supernatant and put it on ice for test.

2. Serum (plasma)mencicipi:

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 larutan standar, dilute to 1, 5, 0.25, 0.125, 0 mol/mL, and use 2, 1, 0.5, 0.25, 0.125, 0 μmol/mL as standard curve.

3. Sample Test

Nama reagen (μL)	Tabung reaksi(At)	Tabung kontrol(Ac)	Tabung standar(As)
Sampel	10	10	–
Solusi standar	–	–	10
Reagen I	50	50	50
Reagen II	10	–	–
Air sulingan	–	10	10
Mixed thoroughly, incubate at 37℃(mamalia) atau 25℃(spesies lain) mandi air untuk 15 menit.
Reagen III	50	50	50
Mixed thoroughly, incubate at 37℃(mamalia) atau 25℃(spesies lain) mandi air untuk 15 menit.
Reagen IV	150	150	150

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, mol/mL, x-axis as the 450 nm absorption. Put ΔA(X) into standard curve, calculate y(mol/mL)
3. Serum (plasma) sample LDH activity

Definisi satuan: 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:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, setiap miligram protein.

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

B. Berat sampel

Definisi satuan: 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. Jumlah sel

Definisi satuan: 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 sel.

LDH(U/104 cell)= y÷T÷500×103 =0.133×y

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

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

500: Jumlah total bakteri atau sel, 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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