实验概要
We provide IHC and WB tips and procedure using alkaline phosphatase to determine if the antibody binds only phosphorylated forms of the target protein.
实验步骤
1. Tissue sections or PVDF membranes (after protein transfer and rehydratation 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).
2. Sister sections or membranes should be incubated in the NE buffer only. Rinse three times in PBST (IHC) or TBST (WB) the sections or membranes, respectively.
3. In PBST (IHC) or TBST (WB), membranes in 5% milk.
4. Incubate sections and membranes overnight in primary antibody.
5. Secondary antibodies concentrations and revelation protocol should be performed as usual.
References:
1. European Journal of Neuroscience, (2005) 21 (7) pp. 1785–97, Sophie Pezet, Achillefs Spyropoulos, Robert J. Williams and Stephen B. McMahon.
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