Chunk #36 — Methods — Western blotting

Source

The Oligodendrocyte Transcription Factor 2 OLIG2 regulates transcriptional repression during myelinogenesis in rodents.

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Cultured cells or mouse brain tissues were homogenized in chilled RIPA lysis buffer (Thermo Fisher Scientific, Cat#89900) for 3 min with homogenate machine, and subsequently centrifuged at 13,000 g for 10 min at 4 °C centrifugal machine, and transferred to a clean 1.5 ml tube. Sample containing 3 g brain tissues were homogenized in chilled RIPA lysis buffer (Thermo Fisher Scientific, Cat#89900) for 3 min with homogenizer machine. Protein was transferred into PVDF membrane (EMD-Millipore, Cat#IPVH00010) after running SDS-PAGE gels in an ice-cold buffer. Immunoblots were probed with indicated antibodies. Primary antibodies include: rabbit anti-SETDB1 (Proteintech, 1:3000, Cat#11231-1-AP), rabbit anti-OLIG2 (talent biomedical, 1:3000, Cat#AP0337), mouse anti-HA probe (Santa Cruz Biotechnology, 1:10000, Cat#SC-7392), rabbit anti-PDGFRα (Santa Cruz Biotechnology, 1:3000, Cat#sc338), mouse anti-GFAP (talent biomedical, 1:3000, Cat#AM0123), rabbit anti-IBA1 (WAKO, 1:3000, Cat#019-19741), mouse anti-NeuN (EMD Millipore, 1:3000, Cat#mab377), mouse anti-CNP (EMD Millipore, 1:3000, Cat#MAB326), goat anti-MBP (Santa Cruz Biotechnology, 1:3000, Cat#sc-13914), mouse anti-GAPDH (Santa Cruz Biotechnology, 1:3000, Cat#365062). Secondary antibodies include: Goat anti-mouse (Thermo Fisher Scientific, 1:5000, Cat#G-21040), goat anti-rabbit (Thermo Fisher Scientific, 1:5000, Cat#G-21234), donkey anti-goat (Thermo Fisher Scientific, 1:5000, Cat#A16005).