A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay.
Transcription factors lie at the center of gene regulation, and their identification is crucial to the understanding of transcription and gene expression. Traditionally, the isolation and identification of transcription factors has been a long and laborious task. We present here a novel method for the identification of DNA-binding proteins seen in electrophoretic mobility shift assay (EMSA) using the power of two-dimensional electrophoresis coupled with mass spectrometry. By coupling SDS-PAGE and isoelectric focusing to EMSA, the molecular mass and pI of a protein complex seen in EMSA were estimated. Candidate proteins were then identified on a two-dimensional array at the predetermined pI and molecular mass coordinates and identified by mass spectrometry. We show here the successful isolation of a functionally relevant transcription factor and validate the identity through EMSA supershift analysis.
Woo
Andrew J
AJ
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences and Western Australian Institute for Medical Research, The University of Western Australia, Crawley 6009, Australia.
Dods
James S
JS
Susanto
Evelyn
E
Ulgiati
Daniela
D
Abraham
Lawrence J
LJ
D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D019169:Jurkat Cells; D040901:Proteomics; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D004247:DNA