Bis tris novax nupage gradient gels

For quantitative Western blots, cells were placed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 10% glycerol) containing phosphatase inhibitor (Thermo Fisher Scientific) and a mixture of protease inhibitors (Roche). Protein lysates (30 μg) were loaded and separated on 4–12% Bis Tris Novax NuPAGE gradient gels (Life Technologies), transferred to nitrocellulose membrane (GE Healthcare). The membrane was dried overnight and incubated in blocking buffer (1× PBS containing 5% non-fat dried milk) for 1 h, followed by incubation with primary antibodies against SOX2, NEUROD1, and ACTB. Primary antibodies were detected using IRDye 800CW or IRDye 680RD conjugated secondary antibodies (1:10,000 dilution) (LI-COR Biosciences). Fluorescence from the membrane was acquired using the Odyssey imaging system (LI-COR Biosciences) and quantified using the Image Studio software (LI-COR Biosciences). Primary antibodies for quantitative Western blotting are listed on Table 2.

Cells were lysed in lysis buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 10% glycerol containing phosphatase inhibitor (Thermo Scientific) and a mixture of protease inhibitors (Roche). Protein lysates (30 μg) were loaded and separated on 4%–12% Bis Tris Novax NuPAGE gradient gels (Life Technologies), transferred to PVDF membrane, and incubated in blocking buffer (phosphate-buffered saline (PBS), 0.1% Tween 20 and 5% nonfat dried milk) for 1 h. To detect proteins of interest, membranes were incubated overnight at 4°C with primary antibodies. Immunoreactive bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies, followed by application of chemiluminescence substrate (Pierce ECL, Thermo Fisher Scientific). Membranes were exposed to either X-ray film (RPI) or Amersham Hyperfilm ECL (GE Healthcare) for signal detection before film development. To detect multiple proteins using the same membrane, membranes were stripped and re-probed with the appropriate primary antibodies. Quantification of the intensity from individual bands was done using Photoshop. Antibodies and dilutions of antibodies for Western blot are described in Table 1.