[An infrared imaging system for detecting electrophoretic mobility shift of DNA-protein complexes].
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To establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides.GILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System.One lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load.This infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.Keywords:
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The electrophoretic mobilities of 34 DNA fragments with a varied phasing of two A-tract bends have been determined in different polyacrylamide gels. They are perfectly described by a cosine function. A systematic variation of the polyacrylamide gel matrix revealed that not only the absolute electrophoretic mobilities of these distance variants, but also their amplitudes and the positions of their extrema, depend on the polyacrylamide content. The displacement of the extrema with increasing polyacrylamide content is attributed to gel-induced unwinding of the DNA. None of these matrix effects can be explained by the change of the pore sizes, which is generally regarded as the central parameter determining electrophoretic mobility of DNA in gels. Instead, we hypothesize an interaction between the electrophoresed DNA and the polyacrylamide matrix and discuss a new qualitative model for electrophoresis of DNA in polyacrylamide gels which accounts for these observations.
Polyacrylamide
Matrix (chemical analysis)
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To establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides.GILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System.One lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load.This infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.
Polyacrylamide
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Journal Article DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in plyacrylamide-gels Get access R. Frank, R. Frank Institut für Organische Chemie und Biochemie, Universität HamburgMartin-Luther-King-Platz 6, D-2000 Hamburg 13, GFR Search for other works by this author on: Oxford Academic PubMed Google Scholar H. Köster H. Köster Institut für Organische Chemie und Biochemie, Universität HamburgMartin-Luther-King-Platz 6, D-2000 Hamburg 13, GFR Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 6, Issue 6, 25 May 1979, Pages 2069–2087, https://doi.org/10.1093/nar/6.6.2069 Published: 01 January 1979 Article history Published: 01 January 1979 Received: 13 March 1979
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We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.
Quantitative Analysis
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Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Electrophoresis moves the non-crosslinked DNA into the gel where the single-stranded product of desired size can be recovered. Our results show this method produces high yields of pure ssDNA.
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In an electrophoretic mobility-shift assay (EMSA, or simply “gel shift”), a 32 P-labeled DNA fragment containing a specific DNA site is incubated with a cognate DNA-binding protein. The protein–DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel. The protein retards the mobility of the DNA fragments to which it binds. Thus, the free DNA will migrate faster than the DNA–protein complex. An image of the gel is used to reveal the positions of the free and bound radiolabeled DNAs.
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The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.
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We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe−target complex (P·T) and unbound P are determined. Distinguishing between P and P·T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P·T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P·T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P·T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P·T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.
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Hybridization probe
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Agarose
Matrix (chemical analysis)
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Protein-DNA interactions were studied on the basis of capillary electrophoretic separation of bound from free fluorescent probe followed by on-line detection with laser-induced fluorescence polarization. Changes in electrophoretic mobility and fluorescence anisotropy upon complex formation were monitored for the determination of binding affinity and stoichiometry. The method was applied to study the interactions of single-stranded DNA binding protein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increases in fluorescence anisotropy and decreases in electrophoretic mobility upon their binding to SSB were observed for the fluorescently labeled 11-mer and 37-mer oligonucleotide probes. Fluorescence anisotropy and electrophoretic mobility were used to determine the binding constants of the SSB with the 11-mer (5 x 10(6) M(-1)) and the 37-mer (23 x 10(6) M(-1)). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multiple protein-DNA complexes that differ in stoichiometry was observed. The results demonstrate the applicability of the method to study complex interactions between protein and DNA.
Binding constant
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