Horseradish peroxidase conjugated anti mouse secondary antibody

The tissue samples were homogenized in 1∶10 (w/v) ice-cold radioimmunoprecipitation assay (RIPA) buffer [in mM, 150 NaCl, 5 EDTA, 1% Triton X-100, 1 Na3VO4, 50 NaF, 1 PMSF, 1 aprotinin, 1 leupeptin, 5 DTT, protease inhibitor cocktail (Sigma-Aldrich P8340), and 10 Tris-Cl, pH 7.4]. The homogenates were centrifuged at 13,000 g for 15 min at 4°C. Supernatants were collected and added to 4× SDS sample buffer. Samples were stored at −20°C until assay and were thawed only once. Mice cortical extracts (100 µg) were electrophoresed on SDS/5% PAGE and transferred to polyvinyldifluoridine membranes. The membranes were incubated overnight at 4°C with the primary antibodies Na_V1.1 (73-023, 1∶200, NeuroMab) and mouse anti Na_V1.2 (73-024,1∶500, NeuroMab) followed by the horseradish peroxidase–conjugated anti-mouse secondary antibody (Amersham Biosciences) 2 h at room temperature. The protein bands were visualized using the Pierce ECL system and scanned. The concentrations of blocking peptides were 1∶20 for Na_V1.1 and 1∶1 for Na_V1.2.

Cells were lysed in RIPA buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS; Thermo Scientific) supplemented with Protease Inhibitor Cocktail (Sigma). An amount of 50 μg of total protein was prepared in 5 × loading buffer supplemented with 10% β-mercaptoethanol and heated for 10 min at 95 °C. Proteins were subjected to SDS–polyacrylamide gel electrophoresisusing 10% polyacrylamide gels. Transfer onto the nitrocellulose membranes was followed by probing with mouse anti-HA antibody (Abcam) at a 1:5,000 dilution. Detection of effector domains was performed with horseradish peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:5,000, followed by incubation with enhanced chemiluminescence (Amersham).