Fueled by the fast-growing genomics information, the next challenge is to characterize protein functionality and understand how cell functions on the systems level. Protein microarrays, especially functional protein microarrays, have become a powerful and versatile tool for proteomics studies. In this seminar, I will first describe the recent technology development in protein microarrays. To illustrate the power of this technology, I will report one of our recent projects to better understand the molecular principle of epigenetic regulation. We used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Finally, in-depth in vivo analysis identified a novel mechanism by which mCpG-dependent binding activity of KLF4 recognizes and activate transcription of genes in closed chromatins. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.