Fig.5

Quantitative benchmarking of the microarray AVEXIS using interactions within the zebrafish Jam family. (A) All six members of the zebrafish Jam family were expressed as biotinylated baits, purified, normalized, serially diluted, and arrayed on streptavidin-coated slides before detection with an anti-Cd4 primary antibody and a Cy3-conjugated goat anti-mouse secondary antibody. The image shows that similar levels of Jam bait proteins were immobilized on the slide. (B) Graphical representation of expected and observed interactions of different affinities within the zebrafish Jam family. The expected (left panel) interactions within the zebrafish Jam family were grouped according to their affinities (red = high affinity [t_1/2 > 2 s]; orange = medium affinity [0.4 < t_1/2 < 2 s]; yellow = low affinity [t_1/2 < 0.4 s]). The observed interactions using the microarray screening approach are depicted graphically in the right panel. (C) The affinity of interactions detected on the microarray correlated with the sensitivity to bait immobilization level in both bait–prey orientations. The ability to detect the four interactions within the Jam family as a function of the immobilized bait level was determined using microarray AVEXIS. Higher affinity interactions (e.g., JamB1–JamC2) were less sensitive to the bait immobilization level, and the same interaction showed the same sensitivity to the bait immobilization level in both bait–prey orientations. RFU, relative fluorescence units. Data points are means ± standard errors (n ⩾ 3).