title: A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.
abstract: We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.
authors: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors: William C
authors: WC
authors: Yang
authors: James R
authors: JR
authors: Swartz
mesh_terms: D001667:Binding, Competitive; D002250:Carbon Radioisotopes / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071317:Nanog Homeobox Protein; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D002250:Carbon Radioisotopes; D005456:Fluorescent Dyes; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D055748:SOXB1 Transcription Factors; C088321:nickel nitrilotriacetic acid; D004247:DNA; D009571:Nitrilotriacetic Acid