ELISA-based Binding Affinity Analysis

ELISAs for binding affinity and specificity were performed as described previously^{17 (link)} with minor modifications. Briefly, biotinylated oligonucleotides were bound to Pierce streptavidin-coated high capacity plates (ThermoFisher) followed by blocking with 3% BSA and incubation with full-length recombinant human GST-tagged UHRF1 (Abnova, catalogue no. H00029128-P01) and DDX24 (Abnova, catalogue no. H00057062-P01), HIS-tagged SMARCA4 (Abcam, catalogue no. ab82237), RBM22 (OriGene, TP760056) and Myc/DDK-tagged DDX1 (OriGene, TP308769) in ELISA buffer (100 mM KCl and 50 mM KH₂PO₄, pH 7.4). After three washes with the ELISA buffer, detection was achieved with an anti-GST HRP (horseradish peroxidase)-conjugated antibody (Abcam, catalogue no. ab3416) diluted to 1:5,000, anti-FLAG HRP-conjugated antibody (Abcam, ab1238,) diluted to 1:15,000 or anti-HIS HRP-conjugated antibody (BioLegend, catalogue no. 652503) diluted to 1:3,000 in an ELISA buffer that contained 3% BSA and 3,3′,5,5′-tetramethylbenzidine ELISA substrate (slow kinetic rate) (Abcam, ab171525). Signal intensity was measured at 450 nm on a SPECTROstar nano microplate reader (BMG Labtech). K_d values were calculated from binding curves assuming a one-site binding model in GraphPad Prism, and standard error of means from three replicates are reported.