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Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells
Author(s) -
Park Soo Hyun,
Ban Eunmi,
Song Eun Joo,
Lee Hyunjung,
Chung Doo Soo,
Yoo Young Sook
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100091
Subject(s) - nfat , biology , electrophoretic mobility shift assay , transcription factor , dna , microbiology and biotechnology , dna binding domain , nucleus , transcription (linguistics) , capillary electrophoresis , biochemistry , gene , linguistics , philosophy
Nuclear factor of activated T‐cells (NFAT) is a transcription factor involved in the development of cardiac and skeletal muscle and the nervous system. NFAT is activated by calcium signal pathway and translocated into the nucleus. The quantification of binding between NFAT and NFAT‐specific DNA gives important information about cardiac hypertrophy. We investigated the binding of NFAT3 in nuclear extracts from H9c2 cells to its specific DNA by capillary electrophoretic mobility shift assay. The binding reaction time required for stable formation of the DNA–NFAT3 complex was 3 h and the separation of the complex and free DNA was achieved within 10 min by CE. The formation of NFAT3–DNA complex was confirmed by the competitive reaction. Comparison of the ratios of complex/free DNA peak area for 1 μM endothelin‐1 (ET‐1)‐treated cells and control cells showed the NFAT3 translocation into the nucleus promoted by ET‐1. The binding constant between NFAT3 and DNA was estimated to be 7.7×10 9 M −1 at 4°C.