PMC:4129400 / 7982-10267 JSONTXT

{"project":"2_test","denotations":[{"id":"25087611-20101245-2046316","span":{"begin":127,"end":129},"obj":"20101245"},{"id":"25087611-22078882-2046316","span":{"begin":127,"end":129},"obj":"22078882"},{"id":"25087611-22562408-2046316","span":{"begin":127,"end":129},"obj":"22562408"},{"id":"25087611-20101245-2046317","span":{"begin":559,"end":561},"obj":"20101245"}],"text":"The first antibody set comprised 296 previously validated antibodies directed against 200 unique cell signaling proteins.21,27,28 The second antibody set represented a completely uncharacterized set of 4,070 antibodies directed against 1,848 unique TFs. Three biological replicates for each of 11–12 individuals were pooled together into 6 pools for screening of these 4,366 rabbit polyclonal antibodies at a 1:1,000 dilution. Printing, gel fabrication, horizontal semidry electrophoresis, transfer, blotting, and scanning were performed as in Ciaccio et al.,21 permitting 96 antibodies to be screened over six pooled lysates per MWA. Antibodies were probed in the 800 channel using goat anti-rabbit IR800-conjugated secondary antibodies (1:5,000) (Invitrogen). A validated mouse monoclonal antibody to β-actin (1:1,500) (Cell Signaling 3700) was included on each blot as a printing control and was measured using a goat anti-mouse Alexa Fluor 680-conjugated secondary antibody (1:7,500) (Invitrogen) in the 700 channel. Fluorescence was quantified using LI-COR Odyssey software (v.3.0) by drawing features around the appropriately sized bands for each sample with a fluorescent protein marker (LI-COR 928-40000) acting as a standard for molecular weight. The raw integrated intensities of each feature were background subtracted using the median of the three pixels surrounding the feature as an estimate of local background, the maximum number of pixels permitted by the LI-COR Odyssey image analysis software to be used for local background estimation. These corrected integrated intensities were used to calculate the average background-corrected integrated intensities of replicate spots. Antibodies that displayed a single predominant band of the predicted size of the targeted protein isoform of interest that accounted for \u003e75% of the entire signal of the lane with a signal-to-noise ratio ≥3 were selected for subsequent population-level quantification by RPPAs; antibodies that displayed at least one band of the predicted size of the targeted protein isoform of interest with a signal-to-noise ratio ≥3 but also additional bands were selected for subsequent population-level quantification by MWAs. Antibodies that passed this screen are listed in Table S2 available online."}