Oct-3/4 Antibody (C-10): sc-5279

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:1000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

For western blot analysis, mESCs were lysed and sonicated in ice-cold S300 buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 0.2% NP-40) and supplemented with protease inhibitors (Roche). For co-immunoprecipitation experiments, cells were lysed in the same lysis buffer. Immunoprecipitation was performed on 1 mg protein extracts using homemade anti-EZH2 (AC22) cross-linked sepharose beads (30 μl slurry for IP, Healthcare, cat. # 170780-01) for 3 to 4 h at 4 °C. Immunocomplexes were washed 5× with S300 buffer and eluted in Laemmli sample buffer. Protein lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. After probing with the suitable primary and secondary antibodies, chemoluminescence signals were captured with the ChemiDoc Imaging System (Bio-Rad).

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