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Star Republic: Guide for Biologists

Protein dephosphorylation

Virtualab Protocols (Chang Bioscience)
1. Divide protein sample into two equal volumes, e.g., 12.5 ml.

2. Add 2.5 ml 10X alkaline phosphatase reaction buffer (1 M NaCl, 100 mM MgCl2, 10 mM DTT, 500 mM Tris-HCl pH7.9).

3. Add 10 ml of calf intestinal alkaline phosphatase (CIP, 10 units/ml) in 50% glycerol and 50 mM KCl, 1 mM MgCl2, 0.1 mM ZnCl2, 10 mM Tris-HCl pH8.2).

4. Incubate at 37 C for 15 minutes.

5. Stop the reaction and analyze results using one of the following methods:

    -SDS-PAGE electrophoresis followed by autoradiography if the proteins are labeled or followed by antibody detection.
    -2D-gel electrophoresis followed by autoradiography if the proteins are labeled or followed by antibody detection.