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Probing concentrati...
Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
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- Frykholm, Karolin, 1977 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg
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- Freitag, Camilla (author)
- Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg
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- Persson, Fredrik, 1979 (author)
- Uppsala universitet,Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg,Beräknings- och systembiologi
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- Tegenfeldt, Jonas O. (author)
- Lund University,Lunds universitet,Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),Fasta tillståndets fysik,Fysiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Solid State Physics,Department of Physics,Departments at LTH,Faculty of Engineering, LTH,University of Gothenburg
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- Graneli, Annette, 1973 (author)
- Gothenburg University,Göteborgs universitet,Institutionen för fysik (GU),Department of Physics (GU),University of Gothenburg
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(creator_code:org_t)
- Elsevier BV, 2013
- 2013
- English.
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 443:2, s. 261-268
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Abstract
Subject headings
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- Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.
Subject headings
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- NATURVETENSKAP -- Fysik -- Den kondenserade materiens fysik (hsv//swe)
- NATURAL SCIENCES -- Physical Sciences -- Condensed Matter Physics (hsv//eng)
Keyword
- Single molecule
- DNA
- Rad51
- Microfluidics
- Supported lipid bilayer
- Fluorescence microscopy
- SUPPORTED LIPID-BILAYERS
- HOMOLOGOUS RECOMBINATION
- STRANDED-DNA
- GRADIENTS
- MECHANISMS
- GENERATION
- DIFFUSION
- FILAMENTS
- GENERATION
Publication and Content Type
- ref (subject category)
- art (subject category)
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