Blocking

1. Rinse the membrane several times with PBS (PBS contains 8 g of NaCl, 0.2 g of KCl 0.2 g, 1.44 g of Na₂HPO₄, 0.24 g of KH₂PO₄ in 1 liter of H₂O. Adjust the pH to 7.2).

2. Immerse the membrane in fresh 5% nonfat dry milk in PBS.

3. Shake at room temperature for 2 hr.

Antibody binding

4. Wash the membrane twice for 5 min each in PBS.

5. Add the primary antibody in fresh 5% nonfat dry milk (10 ml per 15 x 15-cm² blot).

6. Incubate the blot with antibody for at least 1 hr at room temperature with shaking.

7. Wash the blot with four changes of PBS for 5 min each.

8. Add 10 ml per 15 x 15-cm² blot of the alkaline phosphatase-conjugated secondary antibody in fresh 5% nonfat dry milk.

9. Incubate the blot for at least 1 hr at room temperature with shaking.

10. Wash the blot with four changes of PBS for 5 min each.

11. Wash the blot twice in 150 mM NaCl, 50 mM Tris-Cl (pH 7.5).

Detection of alkaline phosphatase activity

12. Prepare the following solutions. These solutions are stable at 4 C for at least 1 year.

NBT: Dissolve 0.5 g of nitroblue tetrazolium in 10 ml of 70% dimethylformamide.
BCIP: Dissolve 0.5 g of bromochloroindolyl phosphate (sodium salt) in 10 ml of 100% dimethylformamide.
Alkaline phosphatase buffer: 100 mM NaCl, 5 mM MgCl2, 100 mM Tris-Cl (pH 9.5).

13. Just prior to developing the blot, prepare fresh substrate solution. Add 66 ul of NBT stock to 10 ml of alkaline phosphatase buffer. Mix well. Add 33 ul of BCIP stock. Use within 1 hr.

14. Add 10 ml of substrate solution per 15 x 15-cm² membrane.

15. When the bands are suitably dark, stop the reaction by rinsing the membrane with PBS containing 20 mM EDTA.

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