Histone Peptide Microarray and Pull-down Assays

Peptide microarray and peptide pull-down assays were performed as described previously^{25 (link)}. Briefly, biotinylated histone peptides were printed in triplicate onto a streptavidin-coated slide (PolyAn) using a VersArray Compact Microarrayer (Bio-Rad). After a short blocking with biotin (Sigma), the slides were incubated with the GST-ENL YEATS domain in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF, 20% fetal bovine serum) overnight at 4°C with gentle agitation. After being washed with the same buffer, the slides were probed with an anti-GST primary antibody and then a fluorescein-conjugated secondary antibody and visualized using a GenePix 4000 scanner (Molecular Devices). For the peptide pull-down assays, 1 μg of biotinylated histone peptides with different modifications were incubated with 1–2 μg of GST-fused proteins in binding buffer (50 mM Tris-HCl 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 hr with rotation. The beads were then washed three times and analyzed using SDS-PAGE and Western blotting.