2.4. Protein Microarray Processing
Protein microarrays were blocked for 30 minutes with DIG Easy Hyb solution (Roche, Basel, Switzerland), washed three times for 5 minutes with 1 × PBS + 0.1% Triton X buffer (PBS 10× pH 7.4 (Life Technologies, Carlsbad, CA, USA), Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and rinsed with ddH2O. Slides were dried for 2 minutes by centrifugation at 900 rpm. Purified IgG samples were adjusted to a concentration of 0.4 μg μL−1 with Melon Gel Purification Buffer and were finally diluted 1:2 with 2 × PBS + 0.2% Triton X buffer containing 6% milk powder. 490 μL of the diluted samples were applied onto a single-chamber hybridization gasket slide (Agilent Technologies, Santa Clara, CA, USA) and the 16k protein slide was placed on top, allowing the spotted protein side of the slide to face the IgG sample. Samples were incubated on the 16k microarray slides for 4 hours under rotating conditions, 12 rpm at room temperature. Slides were washed three times with 1 × PBS + 0.1% Triton X wash buffer and rinsed with ddH2O. As detection antibody, Alexa Fluor® 647 Goat Anti-Human IgG (H+L) (Molecular Probes®, Carlsbad, CA, USA) was used, diluted 1:10,000 in PBS + 0.1% Triton X-100 + 3% milk powder. Incubation was done for 1 hour. Slides were again washed three times with 1 × PBS + 0.1% Triton X washing buffer, rinsed with ddH2O and spin-dried. 
Microarray slides were scanned with an Agilent G2565CA Microarray Scanner System (Agilent Technologies, Santa Clara, CA, USA) using a helium-neon laser (633 nm, red dye channel). Slides were scanned with a resolution (pixel size) of 10 μm and sensitivity level of the red channel photomultiplier tube (PMT) was set to 70%. After scanning, images of slides were loaded into the GenePix® Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA) for subsequent data extraction. Median fluorescent intensity, corrected for local fluorescent background, for each IgG-probed protein spot was exported to a .gpr file for further statistical analysis.