Multiplexed Nucleic Acid Programmable Protein Arrays

Yu, Xiaobo; Song, Lusheng; Petritis, Brianne; Bian, Xiaofang; Wang, Haoyu; Viloria, Jennifer; Park, Jin; Bui, Hoang; Li, Han; Wang, Jie; Liu, Lei; Yang, Liuhui; Duan, Hu; McMurray, David N.; Achkar, Jacqueline M.; Magee, Mitch; Qiu, Ji; LaBaer, Joshua

Each M-NAPPA microarray was blocked with Superblock solution (Pierce, Rockford, IL) for 1 h at 23 °C, briefly washed with water, centrifuged at 1000 rpm for 3 min to dry, and covered with a hybridization chamber (Grace BioLabs, OR). The array was then incubated with 160 μL of human in vitro transcription & translation (IVTT) solution containing human HeLa cell lysate, accessory proteins, reaction mixture, and nuclease-free water (Thermo Fisher Scientific, IL) for 1.5 h at 30 °C and 0.5 h at 15 °C to express the GST-tagged proteins-of-interest. The GST-tagged proteins were displayed on the slide surface via the polyclonal α-GST antibody that was included in the printing mixture. Then, the resulting protein microarray was incubated with 5% (w/v) milk in 1xPBS with 0.2% (v/v) Tween-20 (PBST) for 1 h at 23 °C, followed by three brief washes with PBST. The protein specific antibodies were diluted with 5% milk-PBST at 1:50 or 1:100, respectively, and incubated with the protein microarray for 16 h at 4 °C followed by a 1 h incubation at 23 °C with an Alexa Fluor 555 labeled secondary antibody (Jackson ImmunoResearch Laboratories, PA). After washing three times with PBST, the M-NAPPA slides were briefly rinsed with water and dried by centrifugation (2,000 rpm, 2 min). The arrays were scanned by a Tecan scanner (Männedorf, Switzerland).