Fig.3

Optimization of biotinylated bait immobilization on streptavidin-coated microarrays. (A) A purified biotinylated Cd200R–BLH protein was serially diluted at the indicated concentrations in three different PBS/0.5% BSA-based buffers before being printed using 400-μm-diameter solid pins on streptavidin-coated slides (XanTec). Buffers were based on a PBS/0.5% BSA solution but contained either 0.02% Tween 20 and 1% polyethylene glycol (PEG, average mass = 8 kDa) or 10% glycerol. The buffer containing 0.02% Tween 20 reproducibly immobilized the most protein with the best spot morphology. (B) The top panel shows a representative image of a protein microarray containing a dilution series of six different proteins that were arrayed on a streptavidin-coated slide before being incubated with an anti-rat Cd4 monoclonal antibody followed by an anti-mouse Cy3-conjugated secondary. The bottom panel graphically shows quantitation of the fluorescence intensities from triplicate arrays on the same slide. Biotinylated bait proteins were specifically captured, as shown by saturation of immobilized bait at 400 μg/ml and lack of immobilization of an unbiotinylated control. The biotinylated anti-Cd4 monoclonal antibody (Anti-Cd4-Bio) serves as a positive control. Data points represent the means ± standard errors (n = 3).