Materials

1. Alkaline phosphatase from bovine intestinal mucosa, 2000 U/mg or greater (with 4-nitrophenyl phosphate as substrate); this is usually supplied at a concentration of 10 mg/mL. If ammonium sulfate, Tris-HCl, or any amine is present, it must be removed (see Note 1). There are numerous commercial sources of labeling-grade enzyme.

2. Antibody: this should be the pure IgG fraction or, better, affinity-purified antibody from an antiserum or pure monoclonal antibody (see Chapter 7).

3. Phosphate-buffered saline (PBS): 20 ml sodium phosphate buffer, pH 7.2, containing 0.15 M NaCl.

4. Glutaraldehyde (Note: glutaraldehyde is toxic).

5. 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM MgCl2, 0.02% NaN3 (Note: toxic), and 2% bovine serum albumin (BSA).

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