Ez link maleimide peg2 biotin reagent

A recombinant form of Eap that harbored a single, N-terminal cysteine was expressed and purified from E. coli similarly to wild-type Eap (13 (link)). Eap produced in this manner was site-specifically biotinylated using EZ-link Maleimide-PEG₂-Biotin reagent according to manufacturer's suggestions (ThermoFisher, Inc.). Following derivatization, 2 μg of Eap-biotin were added to samples containing either 20 μl EDTA serum or C4-depleted serum (Complement Technologies), and an appropriate quantity of PBS to give a final volume of 100 μl. The samples were incubated for 1 h at room temperature, after which time 30 μl of 50% (v/v) slurry of magnetic streptavidin-coated Dynabeads were added (Invitrogen, Inc.). Following an additional 15 min incubation, the beads were isolated via a magnet, washed three times with 100 μl of PBS, and all remaining liquid was removed by aspiration. 15 μl of non-reducing Laemmli buffer were added to each sample, and 5 μl of each sample were analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue.

Biotinylated C4b was prepared by overnight, room-temperature incubation of native C4 (1 mg/ml final concentration) with C1s (5 μg/ml final concentration) in the presence of EZ-link Maleimide-PEG₂-Biotin reagent according to manufacturer's suggestions (ThermoFisher, Inc.). The reaction mixture (250 μl in PBS (pH 7.0)) was buffer exchanged into 20 mM tris (pH 8.0), applied to a 1 ml Resource Q anion exchange column (GE LifeSciences), and the bound proteins eluted with a gradient to 1 M NaCl over 10 column volumes. Fractions containing biotinylated-C4b were identified and characterized by a combination of SDS-PAGE and Western blotting using streptavidin-conjugated HRP (ThermoFisher, Inc.). Purified C4b-biotin was pooled, quantified spectrophotometry, and stored at 4 °C in the existing buffer until further use.