Solarbio Kreatinkinase (Ck) Aktivitätstest-Kit

Kreatinkinase (Ck) Aktivitätstest-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Betriebsausrüstung: Spektrophotometer

Katze nein: BC1140

Größe:50T/48S

Komponenten:

Lösung extrahieren: 60 mL ×1. Lagerung bei 4℃.

Reagenz I: Pulver × 1, stored in dark at -20℃. Dissolved in 10 ml destilliertes Wasser vor dem Gebrauch, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagenz II: Pulver × 1, stored at -20℃. Dissolved in 0.5 ml destilliertes Wasser vor dem Gebrauch, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagenz III: powder×2, stored at -20℃. Dissolved in 0.5 ml destilliertes Wasser vor dem Gebrauch, the reagents that cannot be used up shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagenz IV: Pulver × 1, stored at -20℃. Dissolved in 0.65 ml destilliertes Wasser vor dem Gebrauch, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Reagenz V: 15 mL ×1. Lagerung bei 4℃.

 

Produktbeschreibung:

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

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung

Scales, Niedrige Temperaturzentrifuge, Wasserbad konstanter Temperatur, spectrophotometer, 1 mL quartz cuvette and distilled water.

Verfahren

ICH. Extraction of crude enzyme solution:

1. 1. Gewebeprobe:

The proportion of tissue mass (G): volume of Extract solution (ml): 1:5~ 10 (Es wird empfohlen, ungefähr abzuwägen 0.1 G Gewebe, hinzufügen 1 ml Extraktlösung) for ice bath homogeneity. Zentrifuge bei 10000 ×g für 15 Minuten bei 4 ℃, Nehmen Sie den Überstand und legen Sie ihn zum Testen auf Eis.

2. Serumprobe:

Direct determination.

3. Zellprobe:

The number of cells (104): the volume of the Extract solution(ml) ist 500 ~ 1000:1 (1 mL of Extract solution is recommended to be added to 5 Millionen Zellen), the Extract solution is added, and the cells are broken by ultrasonic wave in ice bath (Leistung: 300W, ultrasonic: 3S, Intervall: 7S, total time: 3 Protokoll). Zentrifuge bei, 10000×g für 10 Minuten bei 4 ℃, the supernatant and place it on ice for testing.

II. Test Verfahren:

1. Preheat the spectrophotometer for more than 30 Protokoll, Passen Sie die Wellenlänge an 340 nm, and adjust to zero with distilled water.
2. Funktionierende Lösung: mix Reagent I, Reagenz II, Reagenz III, Reagent IV and Reagent V in the proportion of 70:4:7:10:90 (volume ratio) vor dem Gebrauch. Prepare when the solution will be used. Incubate for 20 minutes in the room temperature before use (this step cannot be omitted).
3. Operation table: add the following reagents into 1 mL cuvette

Reagenzname (μL)	Leeres Rohr (AB)	Testrohr (BEI)
crude enzyme solution	–	200
Funktionierende Lösung	450	450
Destilliertes Wasser	550	350
Add the above reagents into the 1 mL quartz cuvette respectively, mix them well and measure the absorbance value A1 at 340 NM für 10 S, quickly place them in a 37℃ water bath for 3 Protokoll (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-Schutz) = 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 fresh weight) = 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 Zellen.

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: Proteinkonzentration der Probe, mg/ml; W: The mass of sample mass, G;

N: The number of cells, 104 Einheiten; T: Reaktionszeit, 3 Protokoll.

Notiz:

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.

Experimental instances：

1. Take 0.1g of mouse brain, add 1mL of extract solution, homogenate and grind. Take the supernatant, 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, calculate the enzyme activity according to sample weight:

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.

Verweise：

• 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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