Hyaluronan binding of sTMEM2 (Microarray)

Binding of sTMEM2 and the aggrecan G1-link protein complex (positive control) to HA oligomers of different length (dp, degree of polymerisation). The experiment was done using a neoglycolipid-based microarray. Spots were printed to contain 5 fmol of probe. After blocking the slides for 1 h with 2% (w/v) bovine serum albumin (BSA; Merck A7030) in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) containing 5 mM CaCl2, the microarrays were overlaid with sTMEM2 at 300 µg/mL for 90 minutes, followed by 1 h incubation with the detection antibody solution composed of the primary antibody (mouse monoclonal anti-polyhistidine antibody; Merck SAB4200620) precomplexed with the biotinylated anti-mouse IgG (goat polyclonal anti-mouse IgG; Merck B7264; 1:1 ratio at 10 µg/mL, diluent: 1% (w/v) BSA in HBS containing 5 mM CaCl2. Binding was detected with Alexa Fluor-647-labelled streptavidin (Molecular Probes) at 1 µg/mL for 30 minutes. The control HA-binding protein was a native protein isolated from bovine nasal cartilage (complex of aggrecan G1 and link protein; Merck 385911), which was overlaid at 50 µg/mL followed by single step detection with Alexa Fluor-647-labelled streptavidin as mentioned above.