Solarbio Phosphoenolpyruvate Carboxykinase (PEPCK) Kit Uji Aktivitas

Atribut	Detail
Catatan	Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian
Peralatan Operasi	Spektrofotometer
Kucing No	BC3310
Ukuran	50T/48S

Komponen:

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

Reagen I: 45 mL×1, stored at 4℃.

Reagen II: bubuk × 1, stored at -20℃ and protected from light. Dissolve it in 35 ML reagen saya sebelum digunakan. The reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

Reagen III: 45 μL×1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 1:120, to prepare when the solution will be used.

Reagen IV: 155 μL×1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 7:250, to prepare when the solution will be used.

Reagen V: Bedak×1, stored at -20℃ and protected from light. Dissolve it in 2.5 mL air suling sebelum digunakan, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

Deskripsi Produk:

PEPCK (EC 4.1.1.32) banyak ditemukan pada hewan, flowering plants, algae, some fungi, and bacteria. The enzyme catalyzes the conversion of oxaloacetic acid to phosphoenolpyruvate, which is the first-rate limiting enzyme regulating gluconeogenesis.

PEPCK catalyzes oxaloacetic acid to form phosphoenolpyruvate and CO2, pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD+ in turn, and determine the NADH decline rate at 340 nm, which can reflect the PEPCK activity.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan

Spektrofotometer, sentrifuge suhu rendah, water bath pot, 1 mL kuvet kuarsa, pipet yang dapat disesuaikan, mortir/homogenizer, es dan air suling

Prosedur

SAYA. Ekstraksi larutan enzim mentah:

1. 1. Sampel jaringan:

Proporsi massa jaringan (G): volume of extract solution (ml): 1:5~10 (disarankan untuk menimbang 0.1 g tisu, menambahkan 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 ×g untuk 10 menit pada 4 ℃, Ambil supernatan dan letakkan di atas es untuk pengujian.

2. Bacteria or cultured cells:

Pertama, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. Jumlah sel (104): the volume of the extract (ml) adalah 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 juta bakteri atau sel), dan sel -selnya rusak oleh gelombang ultrasonik dalam penangas es (Kekuatan: 200W or 20%, ultrasonik:3S, selang:10S, Mengulang 30 waktu). Then centrifuged at 8000 ×g untuk 10 menit pada 4 ℃, Ambil supernatan dan letakkan di atas es untuk pengujian.

3. Sampel serum: Penentuan langsung.

II. Tes prosedur:

1. Panaskan lebih dulu spektrofotometer lebih dari 30 menit, sesuaikan panjang gelombangnya 340 nm, and adjust to zero with distilled
2. Solusi kerja: mix Reagent II, Reagen III, Reagent IV in the proportion of 7:1:1(V:V:V) sebelum digunakan. Persiapkan saat solusinya akan digunakan.
3. Preheat the working solution at 37℃ (mamalia) atau 25 ℃ (spesies lain) untuk 5 menit.
4. Meja operasi: Add the following reagents to the 1 mL quartz cuvette in turn:

Nama reagen (μL)	Tabung kosong(B)	Tabung reaksi(T)
Sampel	–	50
Air sulingan	50
Solusi kerja	900	900
Reagen V	50	50
Add Reagent V and mix it immediately. Measure the absorbance value A1 at 340 nm for 10s and A2 at 70s. Calculate ΔAT= A1T- A2T, ΔAB= A1B- A2B, and ΔA =ΔAT-ΔAB. Blank tube only needs to be done once or twice.

AKU AKU AKU. Perhitungan PEPCK:

1. Calculation by micro quartz cuvette

• Dihitung dengan konsentrasi protein jaringan:

Definisi aktivitas enzim: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, setiap miligram protein.

PEPCK activity (U/mg keuntungan) = Δa ÷(×d)× VRT × 109 ÷ (Vs × CPR) ÷ T =3215.4×ΔA÷Cpr

• Dihitung dengan kualitas sampel jaringan:

Definisi aktivitas enzim: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, setiap gram sampel.

PEPCK activity (U/g berat segar) = Δa ÷(×d)× VRT × 109 ÷ (Vs ÷ vst × w) ÷T= 3215.4×ΔA÷W

• Dengan jumlah sel:

Definisi aktivitas enzim: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every 10 thousand cells

PEPCK activity (sel U/104)= Δa ÷(×d)× VRV × 109 ÷(VS÷VST×500)÷T=6.43×ΔA

• Dihitung dengan volume serum:

Definisi aktivitas enzim: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every milliliter of serum

PEPCK activity (U/mL) = Δa ÷(×d)×VRV×109÷VS÷T=3215.4×ΔA

e: Molar extinction coefficient of NADH, 6.22× 103 l/mol/cm; D: Diameter cahaya cuvette, 1 cm;

Vrt: Volume total sistem reaksi, 0.001 L;

Vs.: Volume sampel dalam sistem reaksi, 0.05 ml; Vst: Volume solusi ekstrak, 1 ml;

Cpr: Konsentrasi protein sampel, mg/mL, Self-determination of protein concentration; W: Massa massa sampel, G;

T: Waktu reaksi, 1 menit;

500: Jumlah total bakteri atau sel, 5 juta; 109: Unit conversion factor, 1 mol = 109 nmol.

Catatan:

1. When A1 is less than 1 or ΔA is greater than 0.6, it is recommended to dilute the sample to a proper multiple before determination to improve the detection
2. For samples with high enzyme activity, such as animal liver, kidney and other tissues, it is recommended to dilute the extract to 5 times or more for
3. The blank tube is a test well for testing the quality of each reagent component. Under normal conditions, the change does not exceed06.
4. The steps of sample adding and mixing shall be rapid, and the stopwatch timing shall be accurate. Jika kondisinya memungkinkan, it is recommended that two persons cooperate to complete this

Contoh Eksperimental:

1. Take 0.1g liver and add 1 ml extract solution for homogenate. Take the supernatant and dilute it twice with the extract solution. Then operate according to the determination steps. Measure and calculate: ΔAT= A1D- A2D= 1.175-0.532=0.643, ΔAB= A1B – A2B =1.29-1.244=0.046, ΔA = ΔAT – ΔAB = 0.643-0.046 = 0.597

PEPCK activity (massa U/g) = 3215.4×ΔA÷W×10 (rasio pengenceran) = 3215.4×0.597÷0.1×10=191959.4 U/g mass.

2. Take 0.1g aloe vera and add 1 ml extract solution for homogenization, take the supernatant and operate according to the determination steps. Measure and calculate ΔAT = A1T- A2T =1.257-1.106=0.151, ΔAB= A1B- A2B =1.29-1.244=0.046, ΔA = ΔAT – ΔAB =0.151-0.046=0.105

PEPCK activity (massa U/g) = 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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