Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 in Saccharomyces cerevisiae.
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.
Yang
Qian
Q
Medical Systems Biology Research Center, Tsinghua University, Beijing, PR China.
Zhao
Yong-Chao
YC
Xiong
Qiang
Q
Cheng
Jing
J
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*
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors