Atribut	Detail
Catatan	Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian
Peralatan Operasi	Spektrofotometer
Kucing No	BC3230
Ukuran	25T/24S; 50T/48S

Komponen:

Ekstrak larutan: 80ml×1. Penyimpanan pada suhu 4℃.

Reagen 1: 40ml×1. Penyimpanan pada suhu 4℃.

Reagen 2: Bedak×1. Penyimpanan pada -20℃, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. Mencairkan 100 times when used.

Reagen 3: Bedak×1. Penyimpanan pada suhu 4℃, dissolve with 3ml of acetone before use. Reagen 4: 5ml×1. Penyimpanan pada suhu 4℃.

Deskripsi Produk:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, plant, mikroorganisme dan sel yang dikultur. It catalyzes succinic acid to form fumaric acid, reduce FAD to form FADH2. The FADH2 reduce oxidized CoQ to form reduced CoQ, which is a branch of respiratory electron transport chain.

CoQ that a catalytic product of complex II could reduce 2,6-dichloroindophenol, which has absorbance at 605 nm, the activity of enzyme can be calculated by detecting the decrease rate of 2, 6-dichlorindolepheno.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:

Spektrofotometer, mandi air, mesin sentrifugal meja, pipet transfer, 1ml glass cuvette, mortar/ homogenizer, acetone, es dan air suling.

1. Complex II extraction:

• Collecting 0.1g of tissue or 5 juta sel, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 menit. Discard the precipitate and transfer supernatant to another tube, sentrifugasi di, 11000g and 4℃ for 15 menit;
• The supernatant, yaitu, cytoplasmic extract, can be used to determine the complex II leaking from mitochondria, this step can show the effect of mitochondrial extraction;
• Menambahkan 400 μL extraction solution to sediment, splitting with ultrasonication (kekuatan 20%, work time 5s, interval 10 detik, mengulang 15 waktu), used to detect Complex Ⅱ activity and protein content.

Determining step

1. Panaskan spektrofotometer untuk 30 menit, sesuaikan panjang gelombangnya 605 nm, Atur penghitung ke nol dengan suling
2. Sample determination

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 sebelum digunakan. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (sel mamalia) atau 25℃(spesies lain) untuk 15
• Add the following reagents in 1ml glass cuvette:

Nama reagen (ul)	Tabung reaksi (A1)
Sampel	50
Reagen 1	750
Solusi kerja	100
Reagen 4	100
Add the above reagent to the 1ml glass cuvette, aduk rata, detect absorbance at 10s (A1). Put cuvette and react solution together in 37℃(mamalia) atau 25℃(spesies lain) mandi air untuk 2 menit, then take cuvette quickly, dry and detect absorbance for 2 menit (A2), ΔA=A1-A2

Perhitungan:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno per mg of tissue protein in every minute.

Complex Ⅱ Activity (U/mg keuntungan)=[ΔA×Vrv÷(×d)×109]−(Vs×Cpr)÷T =476.2×ΔA÷Cpr

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: light path of cuvette, 1 cm;

Tali: volume reaksi total,1ml; Vs: volume sampel (ml), 0.05 ml;

Cpr: Konsentrasi protein sampel (mg/mL); T: waktu reaksi (menit), 2 menit;

Catatan:

1. Take two or three different samples for prediction before test to ensure the accuracy of experimental results. Dilute supernatant with distilled water if absorbance is higher than 1.5. Dilute sample with distilled water if ΔA>0.4, multiply dilute times in the formula. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use bio surya (PC0020 Protein BCA Perangkat Pengujian). Because protein is contained in the extract, the protein content of the extract itself should be subtracted when determining the protein concentration of the sample.
3. It is recommended to use the sample protein concentration to calculate the enzyme activity. If the sample fresh weight is used to calculate, the enzyme activity of cytoplasmic extract needs to be measured, and the sum of supernatant and precipitation enzyme activity is the total enzyme activity.
4. It’s enough for 50 tube reactions.
5. Lampiran: Berat sampel(50T/24S)

• Supernatan:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)=[ΔA1×Vrv÷(×d)×109]−(W÷Ve×Vs)÷T =476.2×ΔA1÷W ΔA1: supernatant absorbance;

Tali: volume reaksi total,1ml;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: light path of cuvette, 1 cm;

Ve: extract solution volume,1ml; Vs: volume sampel (ml), 0.05 ml; T: waktu reaksi (menit), 2 menit;

W: Berat sampel, G.

• Sediment:

Definisi satuan: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)= [ΔA2×Vrv÷(×d)×109]−(W÷Ve×Vs)÷T =190.5×ΔA2÷W ΔA2: sediment absorbance;

Tali: volume reaksi total,1ml;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: light path of cuvette, 1 cm;

Ve: sediment resuspended volume,0.4 ml; Vs: volume sampel (ml), 0.05 ml;

T: waktu reaksi (menit), 2 menit; W: Berat sampel, G.

• Total activity is the sum of ComplexⅡactivity in supernatant and sediment. Complex Ⅱ(U/g) =476.2×ΔA1÷W+190.5×ΔA2÷W.

Contoh eksperimental:

1. Take 0.1g of rabbit liver sample, menambahkan 1 ML larutan ekstrak, grind and centrifuge the homogenate, and operate according to the determination steps. ΔA1 = A1-A2 = 1.134-1.054 = 0.08 in the supernatant, and ΔA2 =A1-A2 = 1.371-1.347 = 0.024 in the precipitation.

The activity of complex II in the supernatant (massa U/g) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 massa U/g

The activity of complex II in the precipitation (massa U/g) = 190.5×ΔA2÷ W = 190.5×0.024÷0.1 = 45.72

massa U/g

Complex II (massa U/g) = 476.2× ΔA1÷W + 190.5×ΔA2÷W = 476.2×0.08÷0.1 + 190.5×0.024 ÷ 0.1 =426.8U/g mass.

Referensi：

[1] Mühling J, Tiefenbach M, López-Barneo J, dkk. Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart[J]. Journal of molecular and cellular cardiology, 2010, 49(6): 950-961.

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