Solarbio Succinate Dehydrogenase (SDH) Kit za ispitivanje aktivnosti

Succinate Dehydrogenase (SDH) Kit za ispitivanje aktivnosti

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

Detection equipment: Spektrofotometar

Mačka ne: BC0950

Veličina: 50T/48s

Komponente

Reagens i: 60 ml × 1, store at -20℃. Reagens II: 0.6 ml × 1, store at -20℃. Reagens iii: 5 ml × 1, store at 4℃.

Reagens IV: Prah × 1, store at 4℃. When the solution will be used, add it into Reagent III to dissolve for use.

Reagens v: Prah × 1, store at 4℃. Dodati 4 mL of distilled water when the solution will be used, the unused reagents are stored at 4℃.

Reagent VI: Prah × 1, store at -20℃. Dodati 3.333 mL of distilled water when the solution will be used, the unused reagents are stored at 4℃.

Opis

Succinate Dehydrogenase (SDH, EC 1.3.5.1) široko se nalazi kod životinja, biljke, mikroorganizmi i kultivirane stanice. SDH is a marker enzyme of mitochondria, which is a membrane binding enzyme located in the inner membrane of mitochondria. It is also one of the key points of respiratory electron transfer and oxidative phosphorylation. In addition, it provides electrons for the respiratory chain of various prokaryotic cells.

SDH can catalyze the dehydrogenation of succinic acid to fumaric acid. The dehydrogenation can reduce 2,6-dichlorophenol indophenol (DCPIP) under the transfer of phenazine dimethyl sulfate (PMS). 2,6- DCPIP has a characteristic absorption peak at 600 NM. The reduction rate of 2,6-DCPIP is determined by the change of absorbance at 600 NM, which represents the activity of SDH enzyme.

Required but not provided

Spektrofotometar, water-bath, tabletop centrifuge, Podesiva pipeta, minobacač/homogenizator, 1 ML staklena kiveta, led i destilirana voda.

Protocol

Ja. Extraction of SDH:

Accurately weigh 0.1 g of tissue or collect 5 milijun stanica, dodati 1 mL of Reagent I and 10 μL of Reagent II, homogenize by using homogenizer/mortar in ice bath, fully grind, centrifuga na 11000 × g za 10 minutes at 4℃, take the supernatant and place it on ice for testing.

Ii. Postupak

1. Predgrijan spektrofotometar za 30 minuta, prilagoditi valnu duljinu na 600 nm i postavite nulu s destiliranom vodom.
2. Procedure test

Add each reagent to 1 mL glass cuvette in turn, and start timing at the same time of adding Reagent VI, record the initial absorbance A1 at the wavelength of 600 nm for 20 sekundi. Then put the cuvette together with the reaction solution into a water bath of 37℃ (sisavac) ili 25 ℃ (Ostale vrste), and accurate reaction for 1 minuta. Quickly take out the cuvette and dry it, and record the absorbance A2 at 80 seconds at 600 NM. ΔA = A1-A2, obtain ΔAT, Δab.

Iii. Calculation of SDH activity

Calculation formula for determination with 1 ML staklena kiveta.

• Koncentracija proteina

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every milligram tissue protein.

SDH(U/Mg Prot)＝[(ΔAT-ΔAB)÷(ε × D)×VRV×109]÷(Cpr×VS) ÷T＝1555.556×(ΔAT-ΔAB)÷Cpr

• Težina uzorka

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every gram tissue.

SDH(U/g)＝[(ΔAT-ΔAB)÷(ε × D)×VRV×109]÷(VS÷VST×W) ÷T＝1571.111×(ΔAT-ΔAB)÷W

• Germ or cells

Definicija jedinice: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every 10 thousand germ or cells.

SDH(U/104 ćelija)＝[(ΔAT-ΔAB)÷(ε × D)×VRV×109]÷(VS÷VST×500) ÷T

＝3.142×(ΔAT-ΔAB)

VRT: Ukupni volumen reakcije, 0.98×10-3 L;

e.: The molar extinction coefficient of 2,6-DCPIP, 2.1×104 L/mol/cm; d: The light diameter of cuvette, 1 cm;

Vs: Volumen uzorka, 0.03 ml;

Vst: Add the volume of Reagent I and Reagent II, 1.01 ml; T: Vrijeme reakcije(min), 1 minuta;

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

500: Cells or germ, 5 milijun.

Bilješka

1. All reagents and samples shall be placed on ice during the determination to avoid denaturation and deactivation.
2. If ΔA is greater than 0.5, the enzyme solution should be diluted with enzyme extract to obtain ΔA with less than 0.5, which can improve the detection sensitivity.
3. Because the Extract solution contains a certain concentration of protein (oko 1 mg/ml), it is necessary to subtract the protein content of the Extract solution itself when determining the protein concentration of the sample.

Eksperimentalni primjer:

1. Take 0.1g of kidney, dodati 1 mL of Reagent Ⅰ and 10 μL Reagent Ⅱ, grind the homogenate with ice bath, centrifuge at 4℃ and 11000g for 10 min, and place the supernatant on ice. According to the determination procedure, the enzyme activity is calculated as follows: ΔAT= A1T- A2T=0.82-0.681=0.139, ΔAb = a1b- A2B=905-0.904=0.001

SDH activity (U/g masa) = 961.905×(Δat- Δab) ÷ W =2168.13 U/g mass.

Reference

• Fattoretti P, Bertoni-Freddari C, Caselli U, et al. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging[J]. Mechanisms of aging and development, 1998, 101(1-2): 175- 182.

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