Determination of transcription factor isoelectric point by two-dimensional native isoelectric focusing and electrophoretic mobility shift analysis.
A rapid, sensitive, and inexpensive assay is described for the determination of isoelectric points of native transcription factors derived from cell nuclei. This assay depends on a transcription factors' ability to bind DNA with high specificity and obviates the need for specific antisera or additional detection methods in identifying a particular protein following isoelectric focusing. This method has been applied to two ubiquitous proteins, the octamer transcription factor, Oct-1, and the multisubunit CCAAT box factor, NF-Y. Isoelectric points have been determined under native conditions using conventional isoelectric focusing in the first dimension, followed by binding to a specific oligonucleotide DNA probe, and separation of specific DNA/protein complexes from unbound DNA in the second dimension using native polyacrylamide gel electrophoresis. Oct-1 from HeLa cell nuclei was shown to have a pI of 9.6, while NF-Y was shown to have a pI of 4.5. This method is applicable to any transcription factor which binds DNA specifically and may be used to identify changes in surface charge characteristics which occur as a result of alternative splicing events and/or following transcription factor post-translational modification(s).
Bush
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Department of Medicine, University of Colorado Health Sciences Center, Denver 80262.
Currie
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RA
D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D014157:Transcription Factors; D004247:DNA