Alkaline Phosphatase (ALP-A)

Enzymes for Clinical Chemistry

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

Origin	recombinant A. sojae
Systematic name	Orthophosphoric-monoester phosphohydrolase (alkaline optimum)
EC Number	3.1.3.1
Reaction formula	An orthophosphoric monoester + H₂O →→→ An alcohol + Orthophosphate

SPECIFICATION

Appearance	A clear, slightly yellowish solution
Activity	≧9,000 U/mg protein
Protein	10-20 mg, Bradford assay
Storage condition	below -20℃

PROPERTIES

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

APPLICATIONS

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

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 (ΔA_s)
The blank solution is prepared by adding diethanolamine buffer (Reagent B) instead of sample (ΔA₀).