This article discusses the procedure used to validate the phospho-specificity of antibodies for a given experiment.
- The first step is the incubation of PVDF membranes overnight at 37 oC in 1% calf intestinal alkaline phosphatase, diluted in NE buffer. This buffer is composed of NaCl 10 mM, Tris 5 mM (pH 7.9), MgCl2 1 mM and dithiothreitol 0.1 mM. The PVDF membranes are used after protein transfer is complete following rehydration in 100% methanol and then in water.
- Sister membranes are subjected to NE buffer incubation only, and then rinsed three times in TBST.
- The membranes are blocked in a 5% milk TBST solution.
- Following blocking, the membranes are subjected to overnight incubation in the primary antibody.
- The conventional protocol for the detection of the concentrations of secondary antibodies is permformed as usual.
Pezet S, Spyropoulos A, Williams RJ, McMahon SB (2005). European Journal of Neuroscience, 21, 1785–97.
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