FOXP3 Monoclonal Antibody (eBio7979), eBioscience™

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	-Mouse (1:500)

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-Mouse (1:500)

Publication protocol

For the preparation of whole-cell lysates, cells were lysed by whole-cell extract buffer (25 mM HEPES, pH 7.7, 300 mM NaCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1 mM Na3VO4, 50 mM NaF, 0.5 mM dithiothreitol and 10% glycerol) on ice for 30 min. Debris was removed by centrifuge on a Eppendoff microfuge (13,200 r.p.m., 30 min, 4 °C), and protein concentrations in the supernatants quantitated by Bio-Rad protein assay.

For immunoprecipitation, whole-cell lysates were incubated with 1 μg specific antibody overnight at 4 °C. This was followed by incubation with Protein G Mag Sepharose (10 μl; GE Healthcare) for 2 h at 4 °C. The beads were washed five times and analysed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE).

For immunoblots, lysates or immunoprecipitates resolved by SDS–PAGE were transferred to polyvinylidene difluoride membrane (Millipore) with transfer buffer (30 mM Tris, 250 mM glycine, 1 mM EDTA, 20% methanol) at 4 °C for 2 h at 400 mA. Membranes were blocked with 5% non-fat milk in 0.1% TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween-20) at room temperature for 1 h. The membrane was then incubated with primary antibodies overnight at 4 °C. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies in blocking buffer for 1.5 h at room temperature. After washing three times, membranes were developed using ECL western blot detection reagents (GE Healthcare), and signals detected by X-ray film. Immunoblot images have been cropped for presentation.

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