Alkaline Phosphatase (ALP-A) 碱性磷酸酶

  • 临床检测用酶
Alkaline Phosphatase碱性磷酸酶

ALP-A is a highly active alkaline phosphatase. It is produced using genetic recombination technology, so it is an enzyme with stable quality.

由来 recombinant A. sojae
系统名称

Orthophosphoric-monoester phosphohydrolase (alkaline optimum)

EC编号 3.1.3.1
反应式

An orthophosphoric monoester + H₂O →→→ An alcohol + Orthophosphate

规格

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

适用类型

ALP-A is used in immunoassays such as ELISA, immunostaining, and Western blotting by using chromogenic, fluorescent, and chemiluminescent substrates.

ASSAY PROCEDURE

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

  1. 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)
  2. Equilibrate at 37℃ for about 5 min.
  3. Add 0.05 mL of sample and mix.
  4. 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:

EXPERIMENTAL DATA

参考文献



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