Method for detecting transcription factor-protein interactions
View Patent ↗A method is provided for identifying complexes between a transcription factor and another protein, such as another different transcription factor. The method includes the steps of isolating from a biological sample transcription factor complexes based on whether the transcription factor complexes include a particular type of transcription factor; and identifying which of the multiple different proteins are present in the isolated transcription factor complexes.
1. A method for determining the identity of at least one activated transcription factor in a transcription factor-transcription factor complex where the identity of a transcription factor in the complex is known, the method comprising:
contacting a biological sample with a library of double stranded DNA probes under conditions where DNA probe-transcription factor complexes are formed between the DNA probes in the library and activated transcription factors present in the biological sample, the DNA probes each comprising a known recognition sequence varying within the library whereby different DNA probes are capable of binding different activated transcription factors, and the DNA probes in the library being capable of binding to at least two activated transcription factors selected from the group consisting of AP1, AP-2, ARE, Brn-3, C/EBP, CBF, CDP, c-Myb, CREB, E2F-1, EFR, ERE, Ets, Ets-1/PEA3, FAST-1, GAS/ISRE, GATA, GRE, HNF-4, IRF-1, MEF-1, MEF-2, Myc-Max, NF-1, NFATc, NE-E1, NF-E2, NFKB, Oct-1, p53, Pax-5, Pbx1, Pit 1, PPAR, PRE, RAR (DR-5), SIE, Smad SBE, Smad3/4, SP1, SRE, Stat1, Stat3, Stat4, Stat5, Stat6, TFIID, TR, TR (DR-4), USF-1, VDR (DR-3), HSE and MRE;
then contacting the biological sample with an antibody that specifically binds to the transcription factor whose identity is known and isolating the antibody, thereby isolating a DNA probe-transcription factor-transcription factor complex comprising the transcription factor whose identity is known, the at least one transcription factor whose identity is to be determined, and any DNA probes that have bound to the at least one transcription factor whose identity is to be determined;
isolating the DNA probes from the DNA probe-transcription factor-transcription factor complex;
contacting the isolated DNA probes with an array of immobilized hybridization probes under conditions suitable for hybridization of the strands of the DNA probes to the hybridization probes in the array; and
identifying the DNA probes bound to the array, thereby identifying the at least one transcription factor whose identity was to be determined.
2. The method according to claim 1 , wherein the array is an immobilized array.
3. The method according to claim 1 , wherein one strand of the double stranded DNA probes further comprises a detectable marker, the method further comprising detecting the detectable marker to identify which of the isolated DNA probes hybridize to the array.
4. The method according to claim 3 , wherein the detectable marker is biotin.
5. The method according to claim 1 , wherein the sample is a nuclear extract of cells.