Peptide Microarray Binding Assay

The peptide microarrays were generated and assayed as described previously [37 (link), 38 (link)], except that the arrays contained four triplicate spots of each peptide. Briefly, GST-tagged proteins were diluted to 0.5–2 μM in phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween-20 (PBST) and 5% (w/v) bovine serum albumin (BSA, EMD Millipore Omnipure Fraction V) and incubated with peptide microarrays overnight at 4 °C. Arrays were washed three times with PBS and then probed with an anti-GST antibody (EpiCypher Inc.; Cat. No. 13-0022) diluted to 1:1000 in PBST + 5% BSA. Arrays were washed again 3× with PBS and then probed with an Alexa Fluor 647-conjugated anti-rabbit antibody at 1:10,000 (ThermoFisher). Arrays were imaged using a Typhoon Scanner and protein binding was determined as previously described [37 (link), 38 (link)]. The average signal intensity for each peptide was normalized to the most intense binding within an array, and normalized binding was averaged for at least two independent replicates for each protein. Heat maps of relative binding were generated using JavaTree View (version 1.16r4) after normalizing the relative binding within the subset of peptides selected for the heat map.