Conjugates are supplied as a non-azide stabilized solution and are stable for at least 12 months when properly stored. A loss of activity may be observed over the course of several weeks after dilution. Shipped with ice pack for next day delivery.

Concentration:	~1 mg/ml
Storage:	4°C: DO NOT FREEZE

Inhibitors: Inhibitors of alkaline phosphatase activity include organic or inorganic phosphates, chelators of metal ions, and many amino acids (McComb et al, 1979). For this reason the common practice of using phosphate buffered saline as the ELISA buffer should be avoided. Some preparations of p-nitrophenylphosphate are contaminated with enough free phosphate to demonstrably inhibit the enzyme.
 

Detection of ELISA: 1 M Diethanolamine buffer, pH 9.8, containing 1 mg/ml p-nitrophenylphosphate is used. This results in the release of p-nitrophenol which is soluble with an absorption maximum at 410 nm. Alternatively, a substrate which yields an insoluble product may be employed. For example, the following substrate solution may be used:
 

10 ml of 100 mM tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl₂

66 µl of 50 mg/ml nitrobluetetrazolium in 70% dimethylformamide (Stored at 4C)

33 µl of 50 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate in demethylformamide (Stored at 4C) (Blake et al, 1984)

Origin: USA
 

REFERENCES

Blake, M. S., Johnston, K. H., Russell-Jones, G. J. and E. C. Gotschlich. A rapid, sensitive method for detection of alkaline phosphatase-conjugated antibody on Western blots. Analyt. Biochem. 136:175-179 (1984).

Green, N. M. Spectrophotometric determination of avidin and biotin. In Methods in Enzymology, Vol. XVIII (McCormick, D. B. and L. D. Wright, eds.) pg. 418 (1970).

Kincaid, R. L. and M. S. Nightingale. A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling. BioTechniques 6:42-49 (1988).

McComb, R. B., Bowers, G. N. and S. Posen. Alkaline Phosphatase. Plenum Press, New York and London (1979).

Mossner, E., Boll, M. and G. Pfleiderer. Purification of human and bovine alkaline phosphatases by affinity chromatography. Hoppe-Seyler's Z. Physiol. Chem. 361:543 (1980).

O'Shannessey, D. J., Voorstad, P. J. and R. H. Quarles. Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex. Analyt. Biochem. 163:204-209 (1987).

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