Axon genepix array scanner

Phospho Explorer antibody arrays were used to assess differences in serum protein profiles between the three groups. Protein array assays were performed according to the manufacturer's instructions. Briefly, whole serum was depleted of albumin (see below), diluted in labeling buffer (1:15), and mixed with Biotin/DMF labeling solution. Biotinylated proteins from each sample were then incubated on individual antibody-coated slides, followed by fluorescence labeling with Cy3-streptavidin (GE Healthcare). The slides were scanned on an Axon GenePix Array Scanner (Molecular Devices) to detect bound proteins based on fluorescence intensity. Fluorescence intensity of each protein array was normalized and analyzed using GeneSpring software v. 12.5 (Agilent, Santa Clara, CA, USA).

The glass-slide-based phospho-explorer antibody array was utilised according to the manufacturer’s instructions (Full Moon BioSystems Inc.). ZR-75-1 and MDA-MB-468 cells treated with 50 μM AG-205 and PGRMC1 siRNA transfected were harvested using protein extraction buffer (Full Moon BioSystems Inc.) diluted in labelling buffer and mixed with Biotin/DMF labelling solution. Biotinylated proteins were incubated on individual antibody-coated slides, and were labelled with Cy3-streptavidin (GE Healthcare). To detect fluorescent intensity, the slides were scanned using an Axon GenePix Array Scanner (Molecular Devices). The intensity values obtained were normalised relative to internal controls and analysed.

Cell lysates were obtained from primary bone marrow macrophages treated for 30 min with hFc-FNDC4 or hFc control protein (N=2 per condition). The Phospho Explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 1,318 antibodies. Each of the antibodies has two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. The antibody array experiment was performed by Full Moon Biosystems, according to their established protocols50 (link).
The slides were scanned on an Axon GenePix array scanner, and the images were analysed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). The fluorescence signal (I) of each antibody was obtained from the fluorescence intensity of this antibody spot after subtraction of the blank signal (spot in the absence of antibody). For each phospho-specific antibody, a phosphorylation signal ratio was calculated.