Individual bead-type of Luminex xMAP microspheres (Luminex Corporation, Austin, TX) were coupled separately to capture antibodies against total tyrosine kinases using the following procedure. 100µl Microspheres were washed with 100 µl ddH2O and resuspended in 80 µl 100mM NaH2PO4 pH 6.2. 10 µl 25mg/ml Sulfo-NHS (Pierce) and 10 µl 25mg/ml EDC (Pierce) were added and incubated at 25°C for 20min while shaking to activate the microspheres. The activated beads were washed with 250 µl 1x PBS pH7.4 twice and resuspended in 200 µl 1x PBS. 20 µg capture antibodies in 50 µl 1x PBS were added. The mixtures were incubated at 25°C for two hours while shaking in dark. The antibody-coupled beads were blocked with 500 µl PBS-TBN (0.02% Tween-20, 0.1% BSA and 0.05% NaN3 in 1x PBS pH7.4) at 25°C for 30 minutes while shaking. Finally, the microspheres were washed with 500 µl PBS-TBN twice and stored in 500 µl PBS-TBN at 4°C until use. 0.25 µl coupled microspheres per bead type were used for each sample. Microsphere mixture were transferred into a 96-well 1.2um filter plate (Millipore) and washed with 100 µl 1x PBS-1% BSA pH7.4 (Sigma) twice. 100 µl 1mg/ml protein lysates were added to each well and incubated at 4°C overnight while shaking. The microspheres were washed with 100µl 1x PBS-1% BSA pH7.4 twice. 50µl 4µg/ml biotin-4G10 (UpState) were added and incubated at 25°C for 30 minutes while shaking. The microspheres were then washed with 100µl 1x PBS-1% BSA pH7.4 twice and then incubated with 50µl 4µg/ml R-phycoerythrin streptavidin conjugates (Molecular Probes) at 25°C for 10 minutes. Finally, the samples were washed with 100µl 1x PBS-1% BSA pH7.4 twice and resuspended in 50µl 1x PBS-1% BSA pH7.4 for analysis. The data were acquired with a Luminex 100 instrument (Luminex Corporation) according to manufacturer’s instructions. The background readings for each capture antibody were obtained using microspheres incubated with 1x cell lysis buffer (Cell Signaling Technology). Values were considered positive if they were 3-fold over the background and represented by log2-transformation of the folds over background. Negative values were threshold to -10. The preprocessed data were converted into .gct files and analyzed with GenePattern 3.0 (The Broad Institute).
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