Solarbio Lactate Dehydrogenase (LDH) Kit za ispitivanje aktivnosti

Lactate Dehydrogenase (LDH) Kit za ispitivanje aktivnosti

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spectrophotometer/microplate reader

Mačka ne: BC0685

Veličina:100T/48s

Komponente:

Otopina ekstrakta: 60 ml × 1. Skladištenje na 4 ℃；

Reagens i: 7 ml × 1. Skladištenje na 4 ℃.

Reagens II: powder×1. Skladištenje na -20 ℃. Radno rješenje: dissolve with 1.3 ML destilirane vode prije upotrebe. It can be divided into tubule after matching, which can be preserved for two weeks at -20℃. Avoid repeated freeze and thaw cycles.

Reagens iii: 7 ml × 1. Skladištenje na 4 ℃.

Reagens IV: 25 ml × 1. Skladištenje na 4 ℃.

Sodium pyruvate standard solution: 1 ml (2 μmol/ml) ×1. Skladištenje na 4 ℃.

Opis proizvoda:

Lactate dehydrogenase (LDH or LD) is the terminal enzyme of the glycolysis pathway which is found in nearly all living cells (animals, biljke, 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.

Potrebni reagensi i oprema, ali nisu pruženi:

Spectrophotometer/Microplate reader, thermostat water bath, stolna centrifuga, Podesiva pipeta, micro glass cuvette/96 well flat-bottom plate, minobacač/homogenizator, led, destilirana voda.

Postupak:

Ja. Uzorak preparation:

1. Bacteria, stanice, or tissue sample:

Bakterije ili stanice:

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 otopine ekstrakta). Bacteria and cell is split by ultrasonic (stavljen na led, 200W, Radno vrijeme 3s, Interval 10s, ponoviti za 30 vrijeme). Centrifuga na 8000 rpm 4℃ for 10 minuta, take the supernatant and put it on ice for test.

Tkivo:

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 otopine ekstrakta). Ice bath homogenization. Centrifuga na 8000 rpm and 4℃ for 10 minuta, take the supernatant and put it on ice for test.

2. Serum (plazma)uzorak:

Detect sample directly.

Ii. Postupak:

1. Preheat the Spectrophotometer/Microplate reader 30 minuta, prilagoditi valnu duljinu na 450 NM, Postavite nulu s destiliranom vodom.
2. Sodium pyruvate standard solution: take 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 minuta. uzeti 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.

Iii. LDH Calculations.

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 (plazma) sample LDH activity

Definicija jedinice: 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. Tkivo, bacteria or cultured cells LDH activity

A. Koncentracija proteina:

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme catalyzes the producing of 1 nmol of pyruvic acid per minute, Svaki miligram proteina.

LDH(U/Mg Prot)= y÷T÷Cpr×103 =66.7×y÷Cpr

B. Težina uzorka

Definicija jedinice: 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. Količina ćelije

Definicija jedinice: 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 stanice.

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: Vrijeme reakcije, 15 minuta;

CPR: Koncentracija proteina uzorka, mg/ml; W: Težina uzorka, 0.1 g;

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

Reference

[1] Huang P H, Fu L C, Huang C S, et al. 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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