Abstract
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.
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Acknowledgements
We greatly thank Satoko Matsunaga, Yuko Matsubara, Yoshiko Kodani, Yoshinori Tanaka, and two researchers (Mihoro Saeki and Dr. Ryo Morishita) working in CellFree Sciences Co., Ltd for their expert technical assistance. This work was partially supported by the Special Coordination Funds for Promoting Science and Technology by the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Sawasaki, T., Endo, Y. (2010). Cell-Free-Based Protein Microarray Technology Using Agarose/DNA Microplate. In: Endo, Y., Takai, K., Ueda, T. (eds) Cell-Free Protein Production. Methods in Molecular Biology, vol 607. Humana Press. https://doi.org/10.1007/978-1-60327-331-2_7
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DOI: https://doi.org/10.1007/978-1-60327-331-2_7
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