Extracellular Matrix Ligand Binding Assay

In the initial screening assay, binding of the rBucl8-CL-Ct to different
extracellular matrix (ECM) ligands was assessed by ELISA [31 (link)]. Wells were coated overnight with 1 μg
of each ligand dissolved in bicarbonate buffer: collagen type I and IV (Sigma),
elastin (Sigma), fibrinogen (Enzyme Research), plasma fibronectin (Sigma),
cellular fibronectin (Sigma), laminin (Gibco), and vitronectin (Sigma). Next, 1
μg per well of rBucl8-CL-Ct in TBS, 1% BSA was added and incubated for two hours
at 37°C. Wells were washed with TBS and bound rBucl8-CL-Ct was detected with
anti-6His-tag mouse mAb (Proteintech) in TBS-1% BSA and a secondary goat
anti-mouse HRP-conjugated Ab (Jackson Immuno Research Laboratories Inc.);
immunoreactivity was detected with ABTS substrate and measured
spectrophotometrically at OD₄₁₅. Data represent the mean ±SE of three
independent experiments (n = 3), each performed in triplicate wells.
Concentration-dependent binding was assessed in a similar manner, however with
varying concentrations (0–10 μM) of rBucl8-CL-Ct.