Solarbio Creatina Chinasi (CK) Kit di analisi dell'attività

Creatina chinasi (CK) Kit di analisi dell'attività

Nota: Prendi due o tre campioni diversi per la previsione prima del test.

Attrezzatura operativa: Spettrofotometro

Gatto n: BC1140

Misurare:50T/48S

Componenti:

Estrarre la soluzione: 60 ml×1. Conservazione a 4 ℃.

Reagente I: powder×1, stored in dark at -20℃. Dissolved in 10 mL of distilled water before use, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagente II: powder×1, stored at -20℃. Dissolved in 0.5 mL of distilled water before use, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagente III: powder×2, stored at -20℃. Dissolved in 0.5 mL of distilled water before use, the reagents that cannot be used up shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagente IV: powder×1, stored at -20℃. Dissolved in 0.65 mL of distilled water before use, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagente V: 15 ml×1. Conservazione a 4 ℃.

Descrizione del prodotto:

Creatine kinase (CK) (CE 2.7.3.2) is also known as creatine phosphokinase, which mainly exists in heart, muscle, and brain. It can reversibly catalyze the trans-phosphorus reaction between creatine and ATP. It is an important kinase directly related to cell energy transport, muscle contraction and ATP regeneration.

CK catalyzes creatine phosphate and ADP to generate creatine and ATP, hexokinase catalyzes ATP and glucose to generate glucose-6-phosphate, and glucose-6-phosphate dehydrogenase catalyzes glucose-6- phosphate and NADP+ to generate NADPH, resulting in an increase of 340 nm light absorption value, which is used to express CK enzyme activity.

Reagenti e attrezzature necessari ma non forniti

Scales, low temperature centrifuge, bagnomaria a temperatura costante, spettrofotometro, 1 mL quartz cuvette and distilled water.

Procedura

IO. Extraction of crude enzyme solution:

1. 1. Campione di tessuto:

The proportion of tissue mass (G): volume of Extract solution (ml): 1:5~10 (it is recommended to weigh about 0.1 g of tissue, aggiungere 1 ml di soluzione di estratto) for ice bath homogeneity. Centrifugare a 10000 ×g per 15 minuti a 4 ℃, prendi il surnatante e mettilo sul ghiaccio per il test.

2. Campione di siero:

Direct determination.

3. Cell sample:

The number of cells (104): il volume della soluzione di estrazione(ml) is 500~1000:1 (1 mL of Extract solution is recommended to be added to 5 milioni di cellule), the Extract solution is added, and the cells are broken by ultrasonic wave in ice bath (Energia: 300W, ultrasonic: 3S, interval: 7S, tempo totale: 3 minuti). Centrifugare a, 10000×g per 10 minuti a 4 ℃, the supernatant and place it on ice for testing.

II. Test procedura:

1. Preheat the spectrophotometer for more than 30 minuti, regolare la lunghezza d'onda su 340 nm, and adjust to zero with distilled water.
2. Soluzione funzionante: mix Reagent I, Reagente II, Reagente III, Reagent IV and Reagent V in the proportion of 70:4:7:10:90 (volume ratio) before use. Prepare when the solution will be used. Incubate for 20 minutes in the room temperature before use (this step cannot be omitted).
3. Tavolo operatorio: add the following reagents into 1 mL cuvette

III. Calculation of CK:

• Calculated by tissue protein concentration:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmolof NADPH per minute at 37℃ and pH7.0 every milligram of protein.

CK activity (U/mg prot) = ΔA÷(ε×d)×VRT×109 ÷ (VS× Cpr) ÷ T= 268 ×ΔA÷Cpr

• Calculated by the quality of tissue samples:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 in every gram of sample.

CK activity (U/g peso fresco) = ΔA÷(ε×d)×VRT×109 ÷ (VS÷VST×W) ÷T= 268×ΔA÷W

• Calculated by serum volume:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 in every milliliter of serum.

CK activity (U/ml) = ΔA÷(ε×d)×VRV×109 ÷ VS÷T= 268×ΔA

• By cell count:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 every 10000 cellule.

CK activity (Cella U/104)=ΔA÷(ε×d)×VRV×109÷(VS ÷ VST×N)÷T=268 ×ΔA÷ N

e: Molar extinction coefficient of NADPH, 6.22×103 L/mol/cm; D: Light diameter of cuvette, 1 cm;

VRT: Total volume of reaction system, 0.001 ml;

VS: The volume of sample in reaction system, 0.2 ml; VST: The volume of extract solution, 1 ml;

Cpr: Concentrazione proteica del campione, mg/ml; W: The mass of sample mass, G;

N: The number of cells, 104 unità; T: tempo di reazione, 3 minuti.

Nota:

1. The CK of serum is not stable. The samples are collected and measured as soon as possible. The CK of serum is stable for 24 hours after being stored at 4℃ in dark.
2. The protein content of the sample needs to be determined separately. proteina BCA content determination kit can be used for determination.
3. If the OD value is greater than 0.6, the sample can be diluted properly with the extract solution, and calculation formula can be changed according dilution ratio.
4. ΔAB generally does not exceed 01.

Istanze sperimentali：

1. Take 0.1g of mouse brain, aggiungere 1 ml di soluzione di estratto, homogenate and grind. Prendi il surnatante, then dilute with extract 4 times and detect according to the measured steps. Calculate ΔAT=A2T- A1T=0.638-0.149=0.489, ΔAB=A2B-A1B=0， ΔA=ΔAT-ΔAB=0.489-0=0.489, calcolare l'attività enzimatica in base al peso del campione:

CK activity（ U/g weight） =268×ΔA÷W×4（ dilution ratio） =268×0.489÷0.1×4（ dilution ratio）

=5242.08U/g weight.

2. Take 200μL serum of duck to detect directly, calculate ΔAT=A2T-A1T=0.445-0.423=0.022, ΔAB=A2B- A1B=0, ΔA=ΔAT-ΔAB=0.022-0=0.022, calculate the enzyme activity according to volume of serum:

CK activity（U/mL）=268×ΔA=268×0.022=5.896 U/mL.

Riferimenti：

• Defang Li, Ning Lu, JichunHan, et al.Eriodictyol Attenuates Myocardial Ischemia-Reperfusion Injury through the Activation of JAK2. Frontiers in Immunology. January2018;(IF3.845)
• Xu Y, Meng X, Hou X, et al. A mutant of the ButhusmartensiiKarsch antitumor-analgesic peptide exhibitsreducedinhibition to hNav1. 4 and hNav1. 5 channels while retaining analgesic activity[J].Journal of Biological Chemistry, 2017, 292(44):18270-18280

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