Peptide array and analysis

The peptide array containing 384 histone peptides containing 59 posttranslation modifications in duplicates was analyzed to determine the binding specificity of histone-interacting domains. The peptide array used was modified histone peptide array from Active Motif (13001). The peptide array was blocked in 5% nonfat milk followed by incubation with 25 µM GST-tagged purified protein overnight at 4°C in binding buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, 20% fetal bovine serum (FBS), and 0.1% NP-40. This was followed by washing three times in 1× TBST (10 mM Tris at pH 8.0,150 mM NaCl, 0.1% Tween 20). The array was incubated with GST antibody for 2 h, followed by washing with TBST. The array was developed with 5 mL of SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) for 5 min and images were acquired with an Alpha imager. The analysis was done using Array Analyze software from Active Motif, which determines the cutoff and specificity automatically based on background and signal level.