A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay
| Autor: | Andrew J. Woo, Lawrence J. Abraham, J.S. Dods, Daniela Ulgiati, Evelyn Susanto |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Proteomics
Electrophoretic Mobility Shift Assay Biology Biochemistry Binding Competitive Sensitivity and Specificity Analytical Chemistry Jurkat Cells Transcription (biology) Gene expression Humans Electrophoretic mobility shift assay Electrophoresis Gel Two-Dimensional Isoelectric Point Molecular Biology Transcription factor Regulation of gene expression Molecular mass Isoelectric focusing DNA Molecular biology DNA-Binding Proteins stomatognathic diseases Isoelectric Focusing Transcription Factors |
| Zdroj: | Molecularcellular proteomics : MCP. 1(6) |
| ISSN: | 1535-9476 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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