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

After proteins were expressed on M-NAPPA, the resulting protein arrays were blocked with blocking buffer (1×PBS, 1%Tween 20 and 1% BSA, pH 7.4) for 1 h at 4 o C. In parallel, the query proteins (e.g., Rb1, Jun, Fos, LidA) fused to a HaloTag were produced by incubating 90 ng of DNA in 180 µL human cell-free expression system (Thermo Fisher Scientific, IL) for 2 h at 30 o C. To screen protein-protein interactions, the protein array was incubated with unpurified Rb1-Halo protein in human HeLa lysate for 16 h at 4 o C, and then washed with cold washing buffer (PBS, 5 mM MgCl2, 0.5% Tween20, 1% BSA and 0.5% DTT, pH 7.4 at 4 o C) three times to remove unbound molecules. The arrays were consecutively incubated with a chicken anti-Halo tag antibody (GeneTel, WI) and Alexa Fluor 555 goat anti-chicken secondary antibody (Jackson ImmunoResearch Laboratories, PA) for 2 h at 4 o C. Arrays were washed and dried with brief centrifugation at 1,000 rpm for 1 min, and scanned as described above. The protein binding signal was quantified using Array-Pro Analyzer (Media Cybernetics) software as previously reported [20, 33] .