Piruvato carbossilasi (computer) Kit di analisi dell'attività
Nota: Prendi due o tre campioni diversi per la previsione prima del test.
Attrezzatura operativa: Spettrofotometro
Gatto n: BC0730
Misurare:50T/48S
Componenti:
Estrarre la soluzione: Liquido 110 ml×1. Conservazione a 4 ℃.
Reagente I: Liquido 30 ml×1. Conservazione a 4 ℃.
Reagente II: Liquido 10 ml×1. Conservazione a 4 ℃.
Reagente III: Polvere×1. Storage at -20℃. Dissolve with 5 mL of distilled water, store at -20℃ after prepared.
Reagente IV: Polvere×1. Storage at -20℃. Dissolve with 5 mL of distilled water, store at -20℃ after prepared.
Reagente V: Liquido 5 ml×1. Conservazione a 4 ℃.
Reagente VI: Liquido 15 μL×1. Conservazione a 4 ℃.
Reagent VI Diluent Solution: Liquido 10 ml×1. Conservazione a 4 ℃.
Descrizione del prodotto:
Pyruvate carboxylase (computer, CE 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.
Reagenti e attrezzature necessari ma non forniti:
Ultraviolet spectrophotometer, bagnomaria, centrifuga da tavolo, bagnomaria, pipetta regolabile, 1 Cuvetta in quarzo da ml, mortaio/omogeneizzatore, ghiaccio e acqua distillata.
Procedura:
IO. Complex extraction:
- Collecting 0.1 g of tissue or 5 milioni di cellule, aggiungere 1 ml di soluzione di estratto, grinding on ice with mortar/homogenizer.
- Centrifugare a 1000 ×g per 10 minutes at4℃,
- Take the supernatant to other tube and centrifuge at 11000 ×g per 15 minutes at4℃.
- The supernatant is used to detect PC that leaking from mitochondria, which shows the effect of mitochondrial extraction.
- Aggiungere 1 mL of Extract solution to the sediment, splitting with ultrasonic (energia 20%, work time 5s, intervallo 10s, repeat 12 volte), used to detect the enzyme activity of PC and protein content.
II. Determinazione procedura:
- Preheat ultraviolet spectrophotometer for 30 minuti, regolare la lunghezza d'onda su 340 nm, azzerare con acqua distillata.
- Preheat Reagent I at 37℃ for 15 minuti.
- 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.
- Soluzione funzionante: make the solution as the volume ratio of Reagent II: Reagente III: Reagent IV= 2:1:1, prepare the reagent when it will be used.
- Add the following reagents in 1 Cuvetta in quarzo da ml:
| Reagente (μL) | Tubo vuoto (B) | Provetta (T) |
| Reagente I | 450 | 450 |
| Soluzione funzionante | 320 | 320 |
| Reagente V | 80 | 80 |
| Reagente VI | 100 | 100 |
| Campione | – | 50 |
| Acqua distillata | 50 | – |
| Add the above reagents to the 1 mL quartz cuvette in order, timing after add working solution, mescolare accuratamente. Detect the absorbance at 340 nm at the time of 10 secondi, record as AT1 or AB1. Then place dishes with the reaction solution in a 37℃ water bath for 2 minuti. Take it out and wipe it clean, immediately measure the absorbance at the time of 130 secondi, 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. | ||
III. Calcolo:
Definizione di unità: 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 prot)=[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =1607×ΔA÷Cpr
e: NADH molar extinction coefficient, 6.22×103 L/mol/cm; D: Light path of cuvette, 1 cm;
Corda: Volume totale di reazione,1×10-3 litri; Contro: Volume del campione (ml), 0.05 ml;
Cpr: Concentrazione proteica del campione (mg/ml); T: Tempo di reazione (min), 2 minuti;
109: 1 mol=109 nmol.
Nota:
- 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 minuti) 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 (Di 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) Supernatante:
Definizione di unità: 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 (Peso U/g) =[ΔA1×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA1÷W
ΔA1: Supernatant absorbance;
e: NADH molar extinction coefficient, 6.22×103 L/mol/cm; D: Light path of cuvette, 1 cm;
Corda: Volume totale di reazione,1×10-3 litri; Contro: Volume del campione (ml), 0.05 ml; Ve: Extraction solution, 1 ml;
Cpr: Concentrazione proteica del campione (mg/ml); T: Tempo di reazione (min), 2 minuti;
109: 1 mol=109 nmol;
W: Peso del campione, G.
2) Sediment:
Definizione di unità: 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 (Peso U/g) =[ΔA2×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =1607×ΔA2÷W
ΔA2: Sediment absorbance;
e: NADH molar extinction coefficient, 6.22×103 L/mol/cm; D: Light path of cuvette, 1 cm;
Corda: Volume totale di reazione,1×10-3 litri; Contro: Volume del campione (ml), 0.05 ml;
Ve: Sediment heavy suspension volume, 1 ml; Cpr: Concentrazione proteica del campione (mg/ml); T: Tempo di reazione (min), 2 minuti;
109: 1 mol=109 nmol;
W: Peso del campione, G.
3) Totale attività
Total activity is the sum of PC activity in supernatant and sediment. computer(Peso U/g)=1607×ΔA1÷W+1607×ΔA2÷W.
Esempio sperimentale:
- 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 volte. Poi, measured by microquartzplate according to the determination steps, Supernatante: 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
Supernatante: the activity of PC (Massa U/g) = 1607 × Δ A1 ÷ W × 100 (rapporto di diluizione) = 1607×0.215÷0.1× 100 = 345505 Massa U/g;
Precipitation: the enzyme activity of PC (Massa U/g) = 1607×ΔA2 ÷ W×4 (rapporto di diluizione) = 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.
Riferimenti
[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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