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Protein microarrays are able to investigate diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-affinity protein microarray approach we developed for this work, HaloTag nucleic acid programmable protein assay (HT-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Evaluation of the quality of TF-NAPPA by in vitro pull-down assays found that 63% of interactions were reproducible.By bimolecular fluorescence complementation (BiFC) assays, we observed co-localization and biophysical interactions for 25% of the protein-pairs examined.
The application of HT-NAPPA technology to hormone signal pathways allowed the identification of many novel transcription factor protein interactions, and led to the development of a proteome-wide plant hormone TF interactome network.