Horseradish peroxidase conjugated anti his antibody

E2F-1 peptides were synthesized on cellulose membrane as previously described (21 (link)). Briefly, membranes carrying E2F-1 peptides with modified lysine (acetylation) and arginine (citrullination) residues were blocked with 5% (w/v) bovine serum albumin (BSA) in 0.3% Tween 20 phosphate-buffered saline (PBST) for 8 hours at room temperature (RT) and incubated with 1 μM recombinant His-tagged bromodomain at 4°C O/N. The following day, the membranes were washed with PBST buffer and incubated with horseradish peroxidase–conjugated anti-His antibody (Abcam) in 2.5% BSA in PBST for 1 hour at RT. The membranes were then incubated with Pierce ECL Western Blotting Substrate (Thermo Scientific) for 2 min, and luminescence from the membranes was read and captured using the ImageQuant Las 4000 camera.

ELISA was carried out on Nunc Maxisorp plates (Thermo Scientific) coated with 5 μg/mL neutravidin. The blocking was performed with 0.5% (w/v) BSA in PBS for 2 h at room temperature, and 1 μg of biotinylated linker peptide per well was immobilized. Serial dilutions of hexahistidine-tagged LRP6 domains or Reck (R&D systems) were prepared in ELISA buffer [PBS, 0.5% (w/v) BSA, and 0.05% (v/v) tween 20] and added to the wells. The reaction mixture was incubated for 45 min at room temperature and washed with ELISA buffer. Horseradish peroxidase-conjugated anti-His antibody (Abcam, ab1187) was diluted in ELISA buffer at 1:5,000 and added to the wells for 45 min incubation. Linker peptide-bound LRP6 domains or Reck was detected using TMB peroxidase substrate (Seracare, 2-component system), and the plates were read at 450 nm using SpectraMax M5e (Molecular Devices). For the competition assays with Fab YW210, Fab YW211, or H07 VHH, a constant concentration of LRP6 domains at 3x EC50 (SI Appendix, Table S3) was used. A serial dilution of competitors was added, and the bound His-tagged LRP6 domains were detected as described.