高活性のアルカリホスファターゼです。遺伝子組換え技術を用いて生産しており、品質の安定した酵素です。
| 由来 | recombinant A. sojae |
|---|---|
| 系統名 | Orthophosphoric-monoester phosphohydrolase (alkaline optimum) |
| EC 番号 | 3.1.3.1 |
| 反応式 | An orthophosphoric monoester + H₂O →→→ An alcohol + Orthophosphate |
Alkaline Phosphatase (ALP-A) アルカリホスファターゼ(ALP-A)
- 臨床診断用酵素
仕様
| Appearance | A clear, slightly yellowish solution |
|---|---|
| Activity | ≧9,000 U/mg protein |
| Protein | 10-20 mg, Bradford assay |
| Storage condition | below -20℃ |
特性
| Molecular weight | ca. 123 kDa (SDS-PAGE) |
|---|---|
| pH Optimum | 9.5-10 (Fig. 1) |
| Thermal stability | below 50℃ (Fig. 2) |
| Optimum temperature | 50℃ (Fig. 3) |
| Thermal stability (liquid form)) | stable at 37℃ for at least one week (Fig. 4) |
| Activators | Mg2+, diethanolamine |
| Inhibitors | Inorganic phosphate, EDTA |
アプリケーション
ALP-Aは発色基質・蛍光基質・化学発光基質を用いることで、ELISA・免疫染色・ウエスタンブロッティングなどのイムノアッセイに利用されます。
分析手順
Principle
The appearance of p-nitrophenol is measured spectrophotometrically at 405 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which produces 1 μmol of PNP per min at 37℃ and pH 9.8 under the conditions described below.
Reagents
- MgSO4 solution, 1.0 M: 2.47 g of MgSO4 / 10 mL of distilled water.
- Diethanolamine buffer, 1.0 M; pH 9.8, containing 0.5 mM MgSO4: dissolve 52.9 g of diethanolamine in 400 mL of distilled water and add 0.25 mL of MgSO4 solution (Reagent A). Heat the solution to 37℃, then adjust to pH 9.8 with 2 N HCl and dilute with distilled water to 500 mL. Store in a dark bottle and prepare freshly before measurement.
- p-Nitrophenyl phosphate solution, 0.65 M: 247 mg of p-nitrophenyl phosphate / 1.0 mL of distilled water. Store in a dark bottle and prepare freshly before measurement.
Sample: dilute the enzyme preparation to a volume activity of 0.10-0.20 U/mL in ice-cold diethanolamine buffer (Reagent B) immediately before measurement.
Procedure
- Pipette the following reagents into a cuvette (light path: 1 cm)
2.90 mL Diethanolamine buffer (Reagent B) 0.05 mL p-Nitrophenyl phosphate solution (Reagent C) - Equilibrate at 37℃ for about 5 min.
- Add 0.05 mL of sample and mix.
- Record the increase of absorbance at 405 nm in a spectrophotometer heated at 37℃, and calculate the ΔA per min using the linear portion of the curve (ΔAs)
The blank solution is prepared by adding diethanolamine buffer (Reagent B) instead of sample (ΔA0).
Calculation
Activity can be calculated by using the following formula:
実験データ
参考文献
- HN Fernley and PG Walker (1967) Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity, Biochem. J., 104, 1011-1018
- Te Piao King and Loucia Kochoumian (1979) A comparison of different enzyme-antibody conjugates for enzyme-linked immunosorbent assay, J. Immunol. Methods, 28, 201-210
- Price, C. P. and Newman, D. J., “Principles and Practice of Immunoassay,” Macmillan Press, London, 1997.