Protocol tips
Upstream tips |
Protocol tips |
Downstream tips |
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year. |
-Mouse (1:1000)
-To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for
5 min. Lysates can be aliquoted and stored at -20°C for future use. |
|
Upstream tips |
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year. |
Protocol tips |
-Mouse (1:1000)
-To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for
5 min. Lysates can be aliquoted and stored at -20°C for future use. |
Publication protocol
2 ug acid-extracted histones or 10 ug cell extract were run on a 15% (histones) or 10% (cell extract) SDS-PAGE gel and transferred to nitrocellulose. Membranes were blocked with TBST +5% milk and incubated with primary antibody and secondary antibodies for two hours at room temperature in TBST +1% milk. Primary antibody concentrations were 1∶1000 for H3K27me3 (Active Motif 39155), H3K9me3 (Active Motif 39162), H1 (Active Motif 39707), EZH2 (Cell Signaling 3147) & Tubulin (Abcam 16504). Secondary antibody concentrations were 1∶10,000 (Millipore 12–348 & Abcam 102448). Chemiluminescent detection of HRP was done using the SuperSignal West Dura Extended Duration Substrate (Thermo 34075).
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Manufacturer protocol
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