Determination of transcription factor isoelectric pointby two-dimensional native isoelectric focusing and electrophoretic mobility shift analysis

Abstract

A rapid, sensitive, and inexpensive assay is describedfor 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 p I of 9.6, while NF-Y was shown to have a p I 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).