A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
TransLISA, a novel quantitative, nonradioactive assay for transcription factor DNA-binding analyses
Tekijät: Vuori KA, Ahlskog JK, Sistonen L, Nikinmaa M
Kustantaja: WILEY-BLACKWELL PUBLISHING, INC
Julkaisuvuosi: 2009
Journal: FEBS Journal
Tietokannassa oleva lehden nimi: FEBS JOURNAL
Lehden akronyymi: FEBS J
Vuosikerta: 276
Numero: 24
Aloitussivu: 7366
Lopetussivu: 7374
Sivujen määrä: 9
ISSN: 1742-464X
DOI: https://doi.org/10.1111/j.1742-4658.2009.07446.x
Tiivistelmä
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 any transcription factor. Thus, TransLISA can replace EMSAs and may be used in various applications and research fields where quantitative, cost-effective and large-scale measurements of the DNA-binding activity of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary research, environmental monitoring, and drug discovery.
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 any transcription factor. Thus, TransLISA can replace EMSAs and may be used in various applications and research fields where quantitative, cost-effective and large-scale measurements of the DNA-binding activity of transcription factors are required, including screening of responses in multiple treatments in cellular and molecular biology, evolutionary research, environmental monitoring, and drug discovery.
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