tailieunhanh - Báo cáo khoa học: TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA-binding analyses

Transcription factors are DNA-binding proteins that regulate key biologi-cal processes. Their interactions with DNA are commonly analyzed with gel-based electrophoretic mobility shift assay (EMSA) using radioactively labeled probes. Within various fields of research, there exists an increasing demand to develop assays with faster sample throughput combined with improved sensitivity, increased analytical range, and precise quantification. | TransLISA a novel quantitative nonradioactive assay for transcription factor DNA-binding analyses Kristiina A. Vuori1 Johanna K. Ahlskog2 Lea Sistonen2 and Mikko Nikinmaa1 1 Centre of Excellence in Evolutionary Genetics and Physiology Department of Biology University of Turku Finland 2 Department of Biology Abo Akademi University and Turku Centre for Biotechnology University of Turku and Abo Akademi University Finland Keywords DNA-binding activity HSF1 transcription factor TransLISA Correspondence K. A. Vuori Centre of Excellence in Evolutionary Genetics and Physiology Laboratory of AnimalPhysiology Department of Biology FI-20014 University of Turku Finland Fax 358 23336058 Tel 358 23336263 E-mail Website http Received 5 September 2009 revised 14 October 2009 accepted 19 October 2009 doi Transcription factors are DNA-binding proteins that regulate key biological processes. Their interactions with DNA are commonly analyzed with gel-based electrophoretic mobility shift assay EMSA using radioactively labeled probes. Within various fields of research there exists an increasing demand to develop assays with faster sample throughput combined with improved sensitivity increased analytical range and precise quantification. Here we describe the development and performance of a 384-well plate immunoassay termed TransLISA which is a novel homogeneous assay for rapid and sensitive quantification of the DNA-binding activity of transcription factors in cell and tissue lysates. TransLISA outperforms EMSAs because it eliminates the need to use radioactive chemicals and allows fast and precise quantification of DNA-binding activity of transcription factors from large number of samples simultaneously. We have used TransLISA to demonstrate the DNA-binding activity of heat shock factor 1 representing a well-known model of inductive transcriptional regulatory responses but the method is easily adaptable for the study of