Histone peptide microarray analysis reveals high selectivity for amino-acid sequence and combinatorial PTM states by recombinant reader domains. (A-C) Heat map representation of binding specificity of reader fusions and histone antibodies determined from histone peptide microarrays. Reader fusions were probed on the slide at concentrations of 10 nM to 500 nM and antibodies were used as 1:10,000 to 1:2,000 dilution. (A) Effect of amino-acid sequence on binding. Averaged fluorescence signal intensities were normalized to the range between 0 and 1 by the lowest and the highest values of selected peptides for individual array. (B) Effect of combinatorial PTMs on binding. Averaged signal intensities for peptides with combinatorial PTMs were normalized to those with single modifications of interests as log2 ratio. (C) Correct N-terminal context is required for H3K9me3 binding by ATRX-ADD. The signal intensities of K9me3-containing peptides that covered H3 5 to 17 amino acids were quantified and normalized to the highest signals from individual arrays. ATRX-ADD domain was able to bind H3K9me3 peptides covering H3 1 to 13 aa (A) but not the peptides of H3 5 to 17 aa, whereas the antibody binds to both sequences.