Solid-phase binding assay

Recombinant human COL6A2 (abcam 169,887) or human TNFα (R&D) proteins were pre-tested to determine the optimal concentration for coating plates used in the binding assay, which was 1 µM and 0.26 µM respectively. Bait proteins were bound to Nickel coated plates (15,242; Thermo scientific) with 1-h incubation at room temperature. Unbound proteins were washed off and non-specific binding sites was blocked with 1%BSA/TBS. hCCOL6A2 protein-coated plates were incubated with different concentrations of prey proteins, mTNFα (R&D). In the reverse experiment hTNFα coated plates were incubated with different concentration of hCOL6A2 at 4 °C overnight. Unbound proteins were washed off and binding of the prey proteins to the coated plate was detected using primary antibodies raised against abTNFα (abcam 9,739) or abColVI (70R-CR009X, Fitzgerald), combined with species-matched HRP-conjugated secondary antibodies. Plates were read at 450 nm after HRP chromogenic substrate reaction using the TMB-peroxidase substrate system (KPL, Gaithersburg, MD, USA) according to the manufacturer’s protocol.