Mobility-shift analysis with microfluidics chips.
Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.
D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D046911:Macromolecular Substances; D044085:Microfluidics / Q000379:methods*; D008958:Models, Molecular; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; D004247:DNA