title: Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 in Saccharomyces cerevisiae.
abstract: The study on the interactions of nucleic acids with transcription factor (TF) is critical to understand the gene expression at the molecular level. In the present study, a rapid chip-based capillary electrophoretic mobility shift assay with LIF detection has been developed on a PDMS-quartz chip. We used a simple negative pressure injection procedure to avoid the bias of electrokinetic injection and to allow loading of the high salt buffered sample. We observed signals for Cy3-labelled oligonucleotides with a 2.6% RSD in peak height. The specific binding of TF autonomously replicating sequence-binding factor 1 (Abf1), either recombinant Abf1 or endogenous Abf1 in yeast cell extracts, with Cy3-labelled specific capture dsDNA could be analyzed on the uncoated chip filled with 2% hydroxypropylcellulose sieving matrix within 100 s. The specificity of the TF-DNA complex was confirmed by both competition experiment and supershift assay. The interactions between Abf1 in the range from 0.156 to 80 nM and dsDNA capture molecules were examined and a detection limit of 0.156 nM Abf1 was found. The uncoated chip capillary electrophoretic mobility shift assay method described here demonstrated great potential for fast, qualitative and quantitative analysis of protein-DNA interaction with low consumption of samples.
authors.affiliation: Medical Systems Biology Research Center, Tsinghua University, Beijing, PR China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yang
authors.forename: Yong-Chao
authors.initials: YC
authors.lastname: Zhao
authors.forename: Qiang
authors.initials: Q
authors.lastname: Xiong
authors.forename: Jing
authors.initials: J
authors.lastname: Cheng
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000295:instrumentation* / Q000379:methods; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors