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Reference: Yang Q, et al. (2008) Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 in Saccharomyces cerevisiae. Electrophoresis 29(24):5003-9

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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.

Reference Type
Journal Article
Authors
Yang Q, Zhao YC, Xiong Q, Cheng J
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