规格

特性

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 (ΔA_s)
   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

• 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.