Cell-free-based protein microarray technology using agarose/DNA microplate.
Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. We found that Arabidopsis HY5 protein functions as a novel DNA-binding tag (DBtag), and DBtagged proteins are immobilized and purified on a newly designed agarose/DNA microplate. In this chapter, we demonstrate a protocol for making the DBtag-based protein microarray and will provide protocols for two applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, and (2) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1.
Sawasaki
Tatsuya
T
Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Ehime, Japan.
Endo
Yaeta
Y
D000818:Animals; D029681:Arabidopsis Proteins / Q000378:metabolism; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002474:Cell-Free System; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D040081:Protein Array Analysis; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose; D019311:Staurosporine / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; C109254:HY5 protein, Arabidopsis; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D012685:Sepharose; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D011494:Protein Kinases; D019311:Staurosporine