Solarbio kreatin kinaza (Ck) Kit za ispitivanje aktivnosti

Kreatin kinaza (Ck) Kit za ispitivanje aktivnosti

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar

Mačka ne: BC1140

Veličina:50T/48s

Komponente:

Otopina ekstrakta: 60 mL ×1. Skladištenje na 4 ℃.

Reagens i: powder×1, stored in dark at -20℃. Dissolved in 10 ML destilirane vode prije upotrebe, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagens II: powder×1, stored at -20℃. Dissolved in 0.5 ML destilirane vode prije upotrebe, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagens iii: powder×2, stored at -20℃. Dissolved in 0.5 ML destilirane vode prije upotrebe, the reagents that cannot be used up shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagens IV: powder×1, stored at -20℃. Dissolved in 0.65 ML destilirane vode prije upotrebe, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagens v: 15 mL ×1. Skladištenje na 4 ℃.

 

Opis proizvoda:

Creatine kinase (Ck) (EC 2.7.3.2) is also known as creatine phosphokinase, which mainly exists in heart, mišića, 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.

Potrebni reagensi i oprema, ali nisu pruženi

Scales, low temperature centrifuge, Stalna temperaturna vodena kupka, spectrophotometer, 1 mL quartz cuvette and distilled water.

Postupak

Ja. Extraction of crude enzyme solution:

1. 1. Uzorak tkiva:

The proportion of tissue mass (g): volume of Extract solution (ml): 1:5~ 10 (it is recommended to weigh about 0.1 g tkiva, dodati 1 ML otopine ekstrakta) for ice bath homogeneity. Centrifuga na 10000 × g za 15 minutes at 4℃, take the supernatant and place it on ice for testing.

2. Uzorak seruma:

Direct determination.

3. Cell sample:

The number of cells (104): volumen otopine ekstrakta(ml) is 500~1000:1 (1 mL of Extract solution is recommended to be added to 5 milijun stanica), the Extract solution is added, and the cells are broken by ultrasonic wave in ice bath (Power: 300W, ultrasonic: 3s, interval: 7s, ukupno vrijeme: 3 minuta). Centrifuga na, 10000× g za 10 minutes at 4℃, the supernatant and place it on ice for testing.

Ii. Test postupak:

1. Preheat the spectrophotometer for more than 30 minuta, Podesite valnu duljinu na 340 NM, and adjust to zero with distilled water.
2. Radno rješenje: mix Reagent I, Reagens II, Reagens iii, Reagent IV and Reagent V in the proportion of 70:4:7:10:90 (volume ratio) Prije upotrebe. Prepare when the solution will be used. Incubate for 20 minutes in the room temperature before use (this step cannot be omitted).
3. Operativna tablica: add the following reagents into 1 mL cuvette

Ime reagensa (μL)	Blank Tube (Ab)	Test Tube (NA)
crude enzyme solution	–	200
Radno rješenje	450	450
Destilirana voda	550	350
Dodajte gornje reagense u 1 ML kvarcna kiveta, mix them well and measure the absorbance value A1 at 340 nm for 10 s, quickly place them in a 37℃ water bath for 3 minuta (the temperature controlled microplate reader can be set to 37℃), take out the absorbance value A2 at 190 s and calculate the ΔAT = A2T- A1T, ΔAB= A2B- A1B, ΔA =ΔAT-ΔAB. Blank tube only needs to be done 1–2 times.

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 svježa težina) = 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 stanice.

CK activity (U/104cell)= 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: Koncentracija proteina uzorka, mg/ml; W: The mass of sample mass, g;

N: The number of cells, 104 jedinice; T: vrijeme reakcije, 3 minuta.

Bilješka:

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. BCA protein 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.

Eksperimentalni slučajevi：

1. Take 0.1g of mouse brain, Dodajte 1ml otopine ekstrakta, homogenate and grind. Uzmi supernatanta, 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, Izračunajte aktivnost enzima prema težini uzorka:

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.

Reference：

• 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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