Solarbio Laktatdehydrogenase (LDH) Aktivitätstest-Kit

Laktatdehydrogenase (LDH) Aktivitätstest-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Betriebsausrüstung: Spektrophotometer/Mikroplatten-Reader

Katze nein: BC0685

Größe:100T/48S

Komponenten:

Extract solution: 60 ml×1. Lagerung bei 4℃；

Reagenz I: 7 ml×1. Lagerung bei 4℃.

Reagenz II: powder×1. Storage at -20℃. Funktionierende Lösung: dissolve with 1.3 mL of distilled water before use. It can be divided into tubule after matching, which can be preserved for two weeks at -20℃. Avoid repeated freeze and thaw cycles.

Reagenz III: 7 ml×1. Lagerung bei 4℃.

Reagent IV: 25 ml×1. Lagerung bei 4℃.

Sodium pyruvate standard solution: 1 ml (2 μmol/mL) ×1. Lagerung bei 4℃.

Produktbeschreibung:

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

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung:

Spectrophotometer/Microplate reader, thermostat water bath, desk centrifuge, verstellbare Pipette, Mikroglasküvette/96-Well-Platte mit flachem Boden, Mörtel/Homogenisator, Eis, distilled water.

Verfahren:

ICH. Probe preparation:

1. Bacteria, Zellen, or tissue sample:

Bacteria or cells:

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 (placed on ice, 200W, work time 3s, interval 10s, repeat for 30 mal). Zentrifuge bei 8000 rpm 4℃ for 10 Protokoll, take the supernatant and put it on ice for test.

Gewebe:

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. Zentrifuge bei 8000 rpm and 4℃ for 10 Protokoll, take the supernatant and put it on ice for test.

2. Serum (Plasma)sample:

Detect sample directly.

II. Verfahren:

1. Preheat the Spectrophotometer/Microplate reader 30 Protokoll, Wellenlänge anpassen 450 nm, Mit destilliertem Wasser auf Null stellen.
2. Sodium pyruvate standard solution: nehmen 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 Protokoll. Nehmen 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 Berechnungen.

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. Gewebe, bacteria or cultured cells LDH activity

A. Protein concentration:

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 prot)= y÷T÷Cpr×103 =66.7×y÷Cpr

B. Sample weight

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 Zellen.

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: Reaction time, 15 Protokoll;

Cpr: Sample protein concentration, mg/ml; W: Sample weight, 0.1 G;

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

Verweise

[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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