NCSCs were lysed in radioimmunoprecipitation assay buffer [10 mM tris (pH 8.0), 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM EDTA] supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 0.5 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) for 1 hour at 4°C.

Protein extracts (30 to 50 μg per sample) were supplemented with NuPAGE LDS sample buffer (Thermo Fisher Scientific) and 50 mM dithiothreitol (Thermo Fisher Scientific) and denatured at 95°C for 3 min. Then, extracts were run on a precast NuPAGE 4 to 12% bis-tris Gel (Thermo Fisher Scientific) in NuPAGE MOPS SDS Running Buffer (Thermo Fisher Scientific) and transferred to a 0.45-μm nitrocellulose membrane (GE Healthcare) for 1 hour at 100 V in a buffer containing 20% absolute ethanol and 10% 0.25 M tris base and 1.9 M glycine. The membranes were blocked in TBST [50 mM tris (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] and 5% milk for 1 hour, incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 hour at room temperature. Primary [BAZ1B (Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore)] and secondary antibodies were diluted in TBST and 5% milk. Blots were detected with the ECL Prime Western Blotting Detection Reagents (Sigma-Aldrich) and scanned using the ChemiDoc system (Bio-Rad).

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



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