Alexa fluor 647 conjugated affinipure mouse anti human igg h l

Microarray slides were incubated with purified antibody (20 (link)). Purified IgG (1 μg/ml) and purified IgA (5 μg/ml) were diluted 1/10 in SuperBlock T20 (Tris-buffered saline [TBS]) blocking buffer (Thermo Scientific) and incubated for 1 h at 30°C with the peptide microarray slide. The slides were then washed with 5 ml of TBS buffer–0.1% Tween 20 for 3 min on a shaker at room temperature for 5 washes. Next, the slides were placed in the individual chambers of a Sarstedt Quadriperm dish and incubated with Alexa Fluor 647-conjugated AffiniPure mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories Inc.) and biotin-conjugated anti-monkey IgA (Alpha Diagnostics International) for 1 h at room temperature. The slides were then washed 5 times with TBS buffer–0.1% Tween 20. Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc) was added to the slide, and the slide was incubated in the dark for 1 h at room temperature. The slides were then washed 5 times with TBS buffer–0.1% Tween 20 and 5 times with deionized water. To dry, the slides were placed in a 50-ml Falcon tube and spun at 1,400 rpm for 5 min. A control slide incubated with secondary antibodies alone without sample was also run to determine the background.