Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 inSaccharomyces cerevisiae
| العنوان: | Rapid chip-based capillary electrophoretic mobility shift assay with negative pressure injection for the binding study of transcription factor Abf1 inSaccharomyces cerevisiae |
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| المؤلفون: | Qian Yang, Qiang Xiong, Yong-Chao Zhao, Jing Cheng |
| المصدر: | ELECTROPHORESIS. 29:5003-5009 |
| بيانات النشر: | Wiley, 2008. |
| سنة النشر: | 2008 |
| مصطلحات موضوعية: | Detection limit, Saccharomyces cerevisiae Proteins, Chromatography, biology, Oligonucleotide, Clinical Biochemistry, Saccharomyces cerevisiae, Protein Array Analysis, Electrophoretic Mobility Shift Assay, biology.organism_classification, Biochemistry, Analytical Chemistry, DNA-Binding Proteins, Electrophoresis, Microchip, Matrix (chemical analysis), Electrokinetic phenomena, Electrophoretic mobility shift assay, Transcription factor, Quantitative analysis (chemistry), Protein Binding, Transcription Factors |
| الوصف: | 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. |
| تدمد: | 1522-2683 0173-0835 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0768c8c8245b8cbadd7817dd05f48929 https://doi.org/10.1002/elps.200800218 |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi.dedup.....0768c8c8245b8cbadd7817dd05f48929 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15222683 01730835 |
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