Conjugation

Procedure for Conjugation of Alkaline Phosphatase to Antibodies and Other Proteins

At least five different methods for conjugation of alkaline phosphates to proteins have been described.¹ The most popular method is the one-step glutaraldehyde coupling method.²

Procedure:

1. Combine 10 mg of antibody and 5 mg of high activity alkaline phosphatase (Sigma Cat. No. P0114) in 1 ml.
2. Dialyze against 0.1 M sodium phosphate, pH 6.8, overnight to remove any free amine-containing compounds.
3. While gently stirring in a fume hood, add 0.05 ml of a 1% glutaraldehyde solution. Let stir 5 minutes.
4. Incubate for 3 hours at room temperature, and then add 0.1 ml of 1 M ethanolamine pH 7.0.
5. Incubate for an additional 2 hours at room temperature.
6. Dialyze overnight at 4 °C against three changes of PBS.
7. Centrifuge at 40,000 g for 20 minutes to remove unwanted precipitate.
8. Store clarified supernatant at 4 °C in the presence of 50% glycerol, 1 mM ZnCl₂, 1 mM MgCl₂, and 0.02% sodium azide.

1. O’Sullivan, M.J., and Marks, V., Meth. Enzymol., 73, 147-166 (1981).
2. Harlow, E., and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY: 1988) Chapter 9, p. 349.