Solarbio Ca++Mg++-ATP酶活性測定試劑盒

Ca++鎂++-ATPase Activity Assay Kit

筆記: 測試前取兩個或三個不同的樣本進行預測.

操作設備: 分光光度計

目錄號: BC0960

尺寸: 50T/24S

成分:

試劑一: 液體 30 毫升×1. 4℃保存.

試劑二: 液體 4 毫升×1. 4℃保存.

試劑三: Powder×2. -20℃保存. Dissolve thoroughly with 1 使用前的毫升蒸餾水. The rest reagent can be kept at -20℃ for one week.

試劑四: 液體 2 毫升×1. 4℃保存.

試劑V: 粉末×1. 4℃保存. Dissolve thoroughly with 3 使用前的毫升蒸餾水. 試劑六: 粉末×1. 4℃保存. Dissolve thoroughly with 15 使用前的毫升蒸餾水, can be kept at 4℃ for one week.

Reagent VII: 粉末×1. 4℃保存. Dissolve thoroughly with 15 使用前的毫升蒸餾水, can be kept at 4℃ for one week.

Reagent VIII: 液體 15 毫升×1. Storage at RT.

標準溶液: 液體 1 毫升×1. 10 μmol/mL standard phosphorus liquid, 4℃保存.

0.5 μmol/mL standard phosphorus working solution: Dilute the 10 μmol/mL standard 20 times with distilled water to 0.5 μmol/mL standard. 例如: 添加 1.9 mL 蒸餾水至 0.1 mL of standard, mix thoroughly.

Phosphorus fixing reagent:

Prepare reagents for determining phosphorus content: make solution as the volume ratio of H2O: 試劑六: Reagent VII: Reagent VIII =2:1:1:1, which should be light yellow. It shows lose efficacy if color is changed, phosphorus pollution if color is change to blue. Prepare the reagent when it will be used.

筆記: It is better to use new beakers, glass rods and glass pipettes or disposable plastic ware when making reagent to avoid phosphorus pollution.

產品描述:

Ca++Mg++-ATPase is widely distributed in plants, 動物, microorganisms and cells, which catalyzes the hydrolysis of ATP to form ADP and inorganic phosphorus.

Ca++Mg++-ATPase decomposes ATP to produce ADP and inorganic phosphorus. The activity of ATPase can be detected by measuring the amount of inorganic phosphorus.

需要但未提供的試劑和設備:

分光光度計, 桌上型離心機, 可調式移液器, 水浴, 1 毫升玻璃比色皿, 研缽/均質器, 冰和蒸餾水.

程式:

我. 樣品製備:

1. 細菌或細胞:

Collecting bacteria or cells into a centrifuge tube, centrifugation, and discard supernatant. Suggest add 1mL of Reagent I to 5 million of bacteria or cells. Use ultrasonic to splitting bacteria and cells (置於冰上, ultrasonic power 20%, working time 3 秒, 間隔 10 秒, 重複 30 次). 離心機在 8000 ×g 為 10 minutes at 4℃ and take the supernatant on ice before testing.

2. 組織:

添加 1 mL of Reagent I into 0.1 克組織, 在冰上充分研磨. 離心機在 8000 ×g 為 10 minutes at 4℃ and take the supernatant on ice before testing.

3. 血清: Directly

二. 決心:

1. 預熱分光光度計 30 分分鐘, 調整波長至 660 奈米, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:

3. Determination of phosphorus content, add the following reagents to 1.5 mL EP tube:

充分混合, then place the mix solution in a 40℃water bath for 10 分分鐘. Cooling to room temperature and detect the absorbance at 660 奈米. The blank tube and standard tube just need one or two tubes.

三、. 計算:

1. 血清:

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milliliter of serum.

Ca++Mg++-ATPase (單位/毫升)=Cs×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]×Vrv÷s÷T

=7.5×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]

2. 組織, 細菌, 或者 細胞

• 蛋白質濃度:

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milligram of tissue protein.

Ca++Mg++-ATPase (U/毫克蛋白質)=Cs×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]×Vrv÷(Vs×Cpr)÷T

=7.5×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]÷心肺復甦

• 樣品重量:

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour, every milligram of tissue.

Ca++Mg++-ATPase (單位/克重量)=Cs×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]×Vrv÷(Vs÷V1×W)÷T

=7.5×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]÷W

• bacteria or cells

單位定義: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every 10000 cells or bacteria.

Ca++Mg++-ATPase (U/104細胞 )=Cs×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]×Vrv÷(Vs÷V1×500)÷T

=0.015×[ΔA(時間)-ΔA(C)]÷[ΔA(S)-ΔA(乙)]

Cs: Concentrate of standard tube, 0.5 微摩爾/毫升;

繩索: 總反應體積, 0.5 毫升; VS: 樣品量, 0.2 毫升;

心肺復甦: 樣品蛋白濃度 (毫克/毫升); 時間: 反應時間 (分分鐘), 1/6 小時;

瓦: 樣品重量 (G);

Vl: Volume of reagent I, 1 毫升;

500: The amount of bacteria or cell, 5 百萬.

筆記

1. This kit can detect 24 tubes of Ca++Mg++ -ATPase samples in 50 tubes for each sample need one tube as control.
2. This method has the characteristics of trace, sensitive and rapid. The test tubes used for determination are phosphate-free strictly. Avoiding phosphorus pollution is the key to the success of detection.

實驗範例:

1. Take of pancreas and add 1 mL of Reagent I for ice bath homogenization. After centrifugation at 4℃ for 10 分分鐘, the supernatant is put on the ice and operated according to the determination steps. ΔAT = 0.916-0.389=0.527, ΔAS =0.398-0.004=0.394

Ca++Mg++- ATPase activity (U/克質量) = 7.5 × ΔAT ÷ΔAS ÷ W = 100.32 U/克質量.

2. Take 0.1g of willow and add 1 mL of Reagent Ⅰ for ice bath homogenization. After centrifugation at 4℃ for 10 分分鐘, the supernatant is put on ice and operated according to the determination steps. The ΔAT=0.137-0.124=0.013, and the ΔAS=398-0.004=0.394

Ca + + 鎂 + + – ATPase activity (U/克質量) = 7.5×ΔAT ÷ ΔAS ÷ W = 2.47 U/克質量.

參考

[1] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2008, 1777(4): 362-368.

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