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Removal of phosphate groups from proteins, before or after blotting.
To demonstrate specificity of an antibody for a protein in its phosphorylated state, proteins can be dephosphorylated either before SDS-PAGE or after transfer to a membrane. Dephosphorylated samples should show little or no staining compared with untreated samples.
Calf intestinal alkaline phosphatase (CIP)
CIP buffer: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH to 7.9 at 25ºC
Protease inhibitor cocktail, EDTA-free
For an antibody that only binds its phosphoprotein target when the protein is denatured, treating the membrane with phosphatase post-transfer may be preferable to treating the non-denatured lysate with phosphatase, pre-SDS-PAGE. For a well-controlled comparison, the membrane treated with phosphatase should be a piece cut from a blot of a single gel containing a duplicate lane or lanes.