PVDF membranes (after protein transfer and rehydration of the membrane in 100% methanol followed by water) should be incubated overnight at 37°C in 1% calf intestinal alkaline phosphatase diluted in NE buffer 1 (NaCl 10 mM; Tris 5 mM, pH 7.9; MgCl2 1 mM; dithiothreitol 0.1 mM).
Sister membranes should be incubated in the NE buffer only. Rinse three times in TBST.
Block membranes in 5% milk in TBST.
Incubate membranes overnight in primary antibody.
Secondary antibody concentrations and detection protocol should be performed as usual.
Pezet S, Spyropoulos A, Williams RJ, McMahon SB (2005). European Journal of Neuroscience, 21, 1785–97.