Conjugating Alkaline Phosphatase to Antibodies and Other Proteins

Procedure for Conjugation of Alkaline Phosphatase to Antibodies and Other Proteins

At least five different methods for conjugation of alkaline phosphatases 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 (Cat. No. P0114) in 1 mL PBS (20 mM sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl).
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.

References

1. O'Sullivan M, Marks V. 1981. [9] Methods for the preparation of enzyme-antibody conjugates for use in enzyme immunoassay.147-166. https://doi.org/10.1016/0076-6879(81)73062-3

2. Harlow E, Lane D. 1988. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY).349.