Human FcγRs (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb) and MBL were produced via transient transfection in HEK293 cells, and purified via immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) as described previously (Boesch et al., 2014 (link)). Size and purity of all recombinant proteins was confirmed by SDS-PAGE. Human C1q (Fischer Scientific ICN19139101) was purchased unlabeled and biotinylated according to the procedure described below. Biotinylated lectin detection reagents (SNA, ConA, GNL, MAL, LCA, RCA, PNA, AAL, VVL, and UAE1) were purchased from Vector Laboratories (B-1305, B-1005, B-1045, B-1245, B-1085, B-1075, B-1235, B-1315, B-1065, B-1395).
FcRs were chemically biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce 21331) at a molar ratio of 5 mols biotin per mol of protein. Biotinylation was carried out for 2 h at RT, with a protein concentration of 0.2 mg/mL. Afterwards, excess biotin was removed via 3 rounds of dilution with PBS and concentration using 3 kD cutoff centrifugal filter units (Amicon UFC900396).
Immediately prior to use, the biotinylated FcR was mixed with a 1/4th molar ratio of Streptavadin-PE (Prozyme PJ31S), diluted to a final concentration of 1.0 μg/mL FcγR in Assay Buffer (PBS-1X + 0.1% BSA + 0.05% Tween20), and mixed for 10 min with rotation. After mixing, 1% v/v of 500 μM free biotin was added to completely block any free streptavidin binding sites. Biotinylated lectin reagents were obtained commercially from Vector Laboratories. Lectin tetramers were produced in the same manner as FcRs, except that the dilution buffer was 20 mM Tris pH 8.0 + 0.1 mM Ca ++, Mg ++, Mn ++.
FcRs were chemically biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce 21331) at a molar ratio of 5 mols biotin per mol of protein. Biotinylation was carried out for 2 h at RT, with a protein concentration of 0.2 mg/mL. Afterwards, excess biotin was removed via 3 rounds of dilution with PBS and concentration using 3 kD cutoff centrifugal filter units (Amicon UFC900396).
Immediately prior to use, the biotinylated FcR was mixed with a 1/4th molar ratio of Streptavadin-PE (Prozyme PJ31S), diluted to a final concentration of 1.0 μg/mL FcγR in Assay Buffer (PBS-1X + 0.1% BSA + 0.05% Tween20), and mixed for 10 min with rotation. After mixing, 1% v/v of 500 μM free biotin was added to completely block any free streptavidin binding sites. Biotinylated lectin reagents were obtained commercially from Vector Laboratories. Lectin tetramers were produced in the same manner as FcRs, except that the dilution buffer was 20 mM Tris pH 8.0 + 0.1 mM Ca ++, Mg ++, Mn ++.
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