ソーラーバイオ クレアチンキナーゼ (CK) 活性アッセイキット

クレアチンキナーゼ (CK) 活性アッセイキット

注記: テスト前に予測のために 2 つまたは 3 つの異なるサンプルを取得します.

操作装置: 分光光度計

カタログ番号: BC1140

サイズ:50T/48S

コンポーネント:

抽出液: 60 mL×1. 4℃で保存.

試薬I: powder×1, stored in dark at -20℃. Dissolved in 10 使用前に蒸留水 mL, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

試薬II: powder×1, stored at -20℃. Dissolved in 0.5 使用前に蒸留水 mL, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

試薬Ⅲ: powder×2, stored at -20℃. Dissolved in 0.5 使用前に蒸留水 mL, the reagents that cannot be used up shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

試薬IV: powder×1, stored at -20℃. Dissolved in 0.65 使用前に蒸留水 mL, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

試薬V: 15 mL×1. 4℃で保存.

製品説明:

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

必要だが提供されていない試薬と装置

天秤, 低温遠心分離機, 恒温水槽, spectrophotometer, 1 mL quartz cuvette and distilled water.

手順

私. Extraction of crude enzyme solution:

1. 1. 組織サンプル:

The proportion of tissue mass (g): volume of Extract solution (mL): 1:5~10 (it is recommended to weigh about 0.1 組織グラム, 追加 1 抽出液 mL) for ice bath homogeneity. で遠心分離します。 10000 ×gの場合 15 4℃で5分, 上清を採取し、試験のために氷上に置きます.

2. 血清サンプル:

Direct determination.

3. Cell sample:

The number of cells (104): the volume of the Extract solution(mL) is 500~1000:1 (1 mL of Extract solution is recommended to be added to 5 百万個の細胞), the Extract solution is added, and the cells are broken by ultrasonic wave in ice bath (力: 300W, ultrasonic: 3s, 間隔: 7s, 合計時間: 3 分). で遠心分離します。, 10000×gの場合 10 4℃で5分, the supernatant and place it on ice for testing.

Ⅱ. Test 手順:

1. Preheat the spectrophotometer for more than 30 分, 波長を調整して 340 nm, and adjust to zero with distilled water.
2. 実用的なソリューション: mix Reagent I, 試薬II, 試薬Ⅲ, Reagent IV and Reagent V in the proportion of 70:4:7:10:90 (volume ratio) 使用前に. Prepare when the solution will be used. Incubate for 20 minutes in the room temperature before use (this step cannot be omitted).
3. 操作テーブル: 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プロット) = 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/生重量g) = 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 細胞.

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

e: Molar extinction coefficient of NADPH, 6.22×103L/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;

心肺蘇生法: サンプルタンパク質濃度, mg/mL; W: The mass of sample mass, g;

N: The number of cells, 104 units; T: reaction time, 3 分.

注記:

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プロテイン content determination kit can be used for determination.
3. OD 値が より大きい場合 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.

実験用インスタンス：

1. Take 0.1g of mouse brain, 抽出液1mLを加える, homogenate and grind. 上清を採取する, 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, サンプル重量に応じて酵素活性を計算します:

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.

参考文献：

• 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, 他. 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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