Karboksilase Piruvat (komputer) Kit Uji Aktivitas
Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.
Peralatan Operasi: Spektrofotometer
Kucing No: BC0730
Ukuran:50T/48S
Komponen:
Extract solution: Cairan 110 mL×1. Penyimpanan pada suhu 4℃.
Reagen I: Cairan 30 mL×1. Penyimpanan pada suhu 4℃.
Reagen II: Cairan 10 mL×1. Penyimpanan pada suhu 4℃.
Reagen III: Bedak×1. Storage at -20℃. Larutkan dengan 5 mL of distilled water, store at -20℃ after prepared.
Reagen IV: Bedak×1. Storage at -20℃. Larutkan dengan 5 mL of distilled water, store at -20℃ after prepared.
Reagen V: Cairan 5 mL×1. Penyimpanan pada suhu 4℃.
Reagent VI: Cairan 15 μL×1. Penyimpanan pada suhu 4℃.
Reagent VI Diluent Solution: Cairan 10 mL×1. Penyimpanan pada suhu 4℃.
Deskripsi Produk:
Pyruvate carboxylase (komputer, EC 6.4.1.1) is widely present in mitochondria of animals, molds and yeast, but is not found in plants and most bacteria. PC is the main postreaction for oxaloacetate, and is the first-rate- limiting enzyme in the gluconeogenesis process.
PC irreversibly catalyzes pyruvate, ATP, CO2 and water to oxaloacetate, ADP and Pi, malic dehydrogenase further catalyzes the formation of malic acid and NAD+ from acetoacetic acid and NADH. The enzyme activity of PC can be reflected by detecting the oxidation rate of NADH at 340 nm.
Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:
Spektrofotometer ultraviolet, mandi air, mesin sentrifugal meja, mandi air, pipet yang dapat disesuaikan, 1 mL kuvet kuarsa, mortir/homogenizer, es dan air suling.
Prosedur:
SAYA. Complex extraction:
- Collecting 0.1 g of tissue or 5 million cells, menambahkan 1 mL of Extract solution, grinding on ice with mortar/homogenizer.
- Sentrifugasi di 1000 ×g untuk 10 minutes at4℃,
- Take the supernatant to other tube and centrifuge at 11000 ×g untuk 15 minutes at4℃.
- The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
- Menambahkan 1 mL of Extract solution to the sediment, splitting with ultrasonic (kekuatan 20%, work time 5s, interval 10s, repeat 12 waktu), used to detect the enzyme activity of PC and protein content.
II. Tekad prosedur:
- Preheat ultraviolet spectrophotometer for 30 menit, adjust the wavelength to 340 nm, atur nol dengan air suling.
- Preheat Reagent I at 37℃ for 15 menit.
- Diluent Reagent VI according to the volume ratio of Reagent VI: Reagent VI Diluent Solution = 1.6:660(V:V), prepare the reagent when it will be used.
- Solusi kerja: make the solution as the volume ratio of Reagent II: Reagen III: Reagent IV= 2:1:1, prepare the reagent when it will be used.
- Add the following reagents in 1 mL kuvet kuarsa:
| Reagen (μL) | Tabung kosong (B) | Tabung reaksi (T) |
| Reagen I | 450 | 450 |
| Solusi kerja | 320 | 320 |
| Reagen V | 80 | 80 |
| Reagent VI | 100 | 100 |
| Sampel | – | 50 |
| Air sulingan | 50 | – |
| Add the above reagents to the 1 mL quartz cuvette in order, timing after add working solution, aduk rata. Deteksi serapan pada 340 nm at the time of 10 detik, record as AT1 or AB1. Then place dishes with the reaction solution in a 37℃ water bath for 2 menit. Take it out and wipe it clean, immediately measure the absorbance at the time of 130 detik, which record as AT2 or AB2. ΔAT= AT1- AT2, ΔAB= AB1- AB2, ΔA= ΔAT-ΔAB. The blank tube only need to test once or twice. | ||
AKU AKU AKU. Perhitungan:
Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every milligram of protein.
PC Activity(U/mg keuntungan)=[ΔA×Vrv÷(×d)×109]−(Vs×Cpr)÷T =1607×ΔA÷Cpr
e: Koefisien kepunahan molar NADH, 6.22×103 L/mol/cm; D: Jalur cahaya kuvet, 1 cm;
Tali: Volume reaksi total,1×10-3 L; Vs: Volume sampel (ml), 0.05 ml;
Cpr: Konsentrasi protein sampel (mg/mL); T: Waktu reaksi (menit), 2 menit;
109: 1 mol=109 nmol.
Catatan:
- Take one or two different samples for prediction before test. It is recommended to dilute the crude enzyme solution with the Extract solution before the determination if the ΔA>0.8(if measuring with 96 well flat-bottom UV plate, ΔA>0.5). While, extending the response time (5 minutes or 10 menit) if ΔA <0.01.
- The blank tube is a detection hole for detecting the quality of each reagent component, and normally that the change of ΔAB does not exceed 0.05.
- The protein concentration of the sample needs to be determined by yourself. Since the Extract solution contains a relatively high protein concentration (tentang 1 mg/mL), the protein concentration of the Extract solution must be deducted when measuring the protein concentration of the sample.
- 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.
- Reagents in this kit are sufficient to complete 50 tube reactions.
- Appendix: calculation formula of sample weight: (sample test number is50T/24S)
1) Supernatan:
Unit definition: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.
PC Activity (berat U/g) =[ΔA1×Vrv÷(×d)×109]−(W÷Ve×Vs)÷T =1607×ΔA1÷W
ΔA1: Supernatant absorbance;
e: Koefisien kepunahan molar NADH, 6.22×103 L/mol/cm; D: Jalur cahaya kuvet, 1 cm;
Tali: Volume reaksi total,1×10-3 L; Vs: Volume sampel (ml), 0.05 ml; Ve: Extraction solution, 1 ml;
Cpr: Konsentrasi protein sampel (mg/mL); T: Waktu reaksi (menit), 2 menit;
109: 1 mol=109 nmol;
W: Berat sampel, G.
2) Sediment:
Unit definition: One unit of enzyme activity is the amount of enzyme catalyzes the production of 1 nmol of NADH per minute every gram of tissue.
PC Activity (berat U/g) =[ΔA2×Vrv÷(×d)×109]−(W÷Ve×Vs)÷T =1607×ΔA2÷W
ΔA2: Sediment absorbance;
e: Koefisien kepunahan molar NADH, 6.22×103 L/mol/cm; D: Jalur cahaya kuvet, 1 cm;
Tali: Volume reaksi total,1×10-3 L; Vs: Volume sampel (ml), 0.05 ml;
Ve: Sediment heavy suspension volume, 1 ml; Cpr: Konsentrasi protein sampel (mg/mL); T: Waktu reaksi (menit), 2 menit;
109: 1 mol=109 nmol;
W: Berat sampel, G.
3) Total activity
Total activity is the sum of PC activity in supernatant and sediment. komputer(berat U/g)=1607×ΔA1÷W+1607×ΔA2÷W.
Contoh eksperimental:
- 1 mL of Extract solution is added to 0.1 g of rabbit heart tissue for homogenization. The supernatant is diluted 100 times with Extract solution, and the precipitation was diluted 4 waktu. Kemudian, measured by microquartzplate according to the determination steps, Supernatan: the ΔAT = A1T – A2T = 1.104-0.856 =0.248, ΔAB = A1B – A2B = 1.021-0.988=0.033, Δ A1 = ΔAT – ΔAB = 0.248-0.033=0.215, precipitate: ΔAT = A1T – A2T = 1.07-0.716 =0.354, ΔAB= A1B- A2B = 1.021-0.988=0.033, ΔA2 = ΔAT- ΔAB =0.354-0.033= 0.321
Supernatan: the activity of PC (massa U/g) = 1607 × Δ A1 ÷ W × 100 (rasio pengenceran) = 1607×0.215÷0.1× 100 = 345505 massa U/g;
Precipitation: the enzyme activity of PC (massa U/g) = 1607×ΔA2 ÷ W×4 (rasio pengenceran) = 1607×0.321÷ 01.
× 4=20633.88 U/g mass;
The total enzyme activity of PC (massa U/g) = 1607×ΔA1÷W×100 (dilution) + 1607×ΔA2 ÷ W
=1607 × 0.215 ÷0.1×100+1607×0.321÷0.1×4=366138.88 U/g mass.
Referensi
[1] Esmail S. Kakey,Amez A. Ismael. Evaluation of Oxidative Stress Status in Aged Human in relation to some Diseases. International Conference on Pure and Applied Sciences. August 2018;
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