Antibody microarray slide

The obtained protein sample (50 ug) was brought to 75 ul with labeling buffer, treated with 3 ul of 10 ug/ul biotin/DMF solution, and incubated at RT for 1 h with mixing. Stop reagent (35 ul) was added, and the sample was incubated at RT for 30 min with mixing. The antibody microarray slide (Fullmoon Biosystems) was shaken (55 rpm) with 30 ml of blocking solution in a petri dish for 1 h at RT. After blocking, the slide was rinsed with Milli-Q grade water. The labeled sample was mixed with 6 ml of coupling solution, and the blocked array slide was shaken with the coupling mixture (60 rpm) for 2 h at RT in a coupling dish. The slide was then washed six times with 30 ml of washing solution in a petri dish with shaking (55 rpm) for 5 minutes, and then rinsed with Milli-Q-grade water. For detection, 30 ul of 0.5 mg/ml Cy3-streptavidin (GE Healthcare, Chalfont St. Giles, UK) was mixed with 30 ml of detection buffer and shaken (55 rpm) with the coupled array slide for 20 minutes at RT. The slide was washed six times with 30 ml of washing solution in a petri dish (55 rpm, 5 minutes each), and rinsed with Milli-Q-grade water.

The protein was extracted from plasma using a protein extraction buffer (Fullmoon biosystems, Sunnyvale, CA) and protein expression was analysed using antibody microarray analysis (Fullmoon biosystems) according to the manufacturer’s protocol. In brief, 50 μg of the protein sample was labelled and incubated with a coupling mixture on the antibody microarray slide (Fullmoon biosystems) and detected with Cy3-streptavidin (GE Healthcare, Chalfont St. Giles, UK). The slide was rinsed and scanned using GenePix 4100 A (Axon Instrument, USA) at a 10 μm resolution, optimal laser power, and PMT. After obtaining the scanned images, they were grided and quantified with GenePix 7.0 Software (Axon Instrument, USA). The data about protein information was annotated using UniProt DB.