Doubly Biotinylated λ Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication"
Article Title: Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication
Figure Legend Snippet: Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.
Techniques Used: Staining, Fluorescence, Migration, Imaging, Concentration Assay
Figure Legend Snippet: Histone dynamics during DNA licensing in HSS. (A) Schematic of the experimental set-up for real-time single-molecule imaging of nucleosome dynamics during replication in Xenopus leavis egg extracts. λ DNA containing fluorescent nucleosomes (one of the four histones labelled fluorescently) is stretched under flow and tethered at both ends to the functionalized glass surface of a microfluidic flow cell. The immobilized DNA is licensed in high-speed supernatant (HSS). Bidirectional replication is initiated upon introduction of nucleoplasmic extract (NPE) supplemented with a fluorescent fusion protein Fen1-KikGR, which decorates replication bubbles and allows progression of replication forks to be tracked in real time. Cy5- or Alexa647-labelled histones within immobilized nucleosomal templates are imaged with a 640-nm laser at each stage. Replication fork progression is visualized in NPE using a 488-nm laser. (B and C) Kymograms and corresponding intensity profiles for fluorescent λ nucleosomes during incubation in HSS. Nucleosomes labelled at H2A-K119C with Cy5 and H2B-T112C with AlexaFluor647 (B) show faster loss of fluorescence than nucleosomes labelled at H3-K36C with Cy5, H3-T80C with AlexaFluor647 and H4-E63C with AlexaFluor647 (C). (D) Plot showing the mean loss of fluorescent signal for λ nucleosomes (H2A-K119 Cy5 , H2B-T112C A647 , H3-K36C Cy5 , H3-T80C A647 and H4-E63C A647 ) during incubation in HSS. Over 100 molecules were analyzed for each histone template. Individual fluorescence decay traces were normalized to background (‘0’) and maximum value of fluorescence (‘1’). A mean fluorescence value and standard deviation were calculated and plotted for each time point. The mean value traces were then fitted to a single exponential function. (E) Summary of the fluorescence decay rate constants ( K ) and half-lives ( t 0.5) extracted from the single exponential fit to the data presented in panel C. See Table S2 for detailed fitting parameters.
Techniques Used: Imaging, Incubation, Fluorescence, Standard Deviation
Figure Legend Snippet: Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (
Techniques Used: SDS Page, Staining, Fluorescence, Electrophoretic Mobility Shift Assay