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a-c GFP translation from localized mRNA-NPs. a , Schematic of the translation from asymmetrically localized mRNA-NPs performed in HeLa cell extract confined in droplets. The HeLa extracts droplets were incubated in an observation chamber beside a permanent magnet. b , Epifluorescence microscopy observation of <t>Cy5</t> labeled mRNAs conjugated to NPs and asymmetrically aligned perpendicularly to the magnet side of the droplet interface. Translation of mRNA-NPs produces GFP that are homogeneously diffusing within the droplet. Scale bar, 20 µm ( b ). c , Relative GFP concentration measured in droplets after 4hr incubation with or without magnet. d-f GFP-mCherry translation from asymmetrically localized mRNA-NPs. d , Top: Schematic of translation process on localized mRNA-NPs: N-terminal nascent GFP are anchored to mRNA-NP via translating mCherry peptides and ribosome. Bottom: Representative time-lapse images of translated GFP and mCherry with epifluorescence microscopy. e , Cy5 labeled mRNA on NPs are colocalizing with translated GFP. f , Time-dependent evolution of the normalized mean fluorescent intensity of GFP and mCherry in selected regions of interested of the droplets (one frame every 5 minutes). Error bars represent standard deviation (SD). g,h, Representative time-lapse images of the translated GFP from localized mRNA-NPs for mRNA(GFP-122a.a) and mRNA(GFP-375a.a.-mCherry), respectively. i, Time dependent evolution of the mean fluorescent intensity of accumulated GFP on mRNA-NPs for mRNA(GFP-375a.a.-mCherry), mRNA(GFP-122a.a) and mRNA(GFP-mCherry).
Cy5, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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a-c GFP translation from localized mRNA-NPs. a , Schematic of the translation from asymmetrically localized mRNA-NPs performed in HeLa cell extract confined in droplets. The HeLa extracts droplets were incubated in an observation chamber beside a permanent magnet. b , Epifluorescence microscopy observation of Cy5 labeled mRNAs conjugated to NPs and asymmetrically aligned perpendicularly to the magnet side of the droplet interface. Translation of mRNA-NPs produces GFP that are homogeneously diffusing within the droplet. Scale bar, 20 µm ( b ). c , Relative GFP concentration measured in droplets after 4hr incubation with or without magnet. d-f GFP-mCherry translation from asymmetrically localized mRNA-NPs. d , Top: Schematic of translation process on localized mRNA-NPs: N-terminal nascent GFP are anchored to mRNA-NP via translating mCherry peptides and ribosome. Bottom: Representative time-lapse images of translated GFP and mCherry with epifluorescence microscopy. e , Cy5 labeled mRNA on NPs are colocalizing with translated GFP. f , Time-dependent evolution of the normalized mean fluorescent intensity of GFP and mCherry in selected regions of interested of the droplets (one frame every 5 minutes). Error bars represent standard deviation (SD). g,h, Representative time-lapse images of the translated GFP from localized mRNA-NPs for mRNA(GFP-122a.a) and mRNA(GFP-375a.a.-mCherry), respectively. i, Time dependent evolution of the mean fluorescent intensity of accumulated GFP on mRNA-NPs for mRNA(GFP-375a.a.-mCherry), mRNA(GFP-122a.a) and mRNA(GFP-mCherry).

Journal: bioRxiv

Article Title: Nanoparticle-based local translation reveals mRNA as translation-coupled scaffold with anchoring function

doi: 10.1101/483727

Figure Lengend Snippet: a-c GFP translation from localized mRNA-NPs. a , Schematic of the translation from asymmetrically localized mRNA-NPs performed in HeLa cell extract confined in droplets. The HeLa extracts droplets were incubated in an observation chamber beside a permanent magnet. b , Epifluorescence microscopy observation of Cy5 labeled mRNAs conjugated to NPs and asymmetrically aligned perpendicularly to the magnet side of the droplet interface. Translation of mRNA-NPs produces GFP that are homogeneously diffusing within the droplet. Scale bar, 20 µm ( b ). c , Relative GFP concentration measured in droplets after 4hr incubation with or without magnet. d-f GFP-mCherry translation from asymmetrically localized mRNA-NPs. d , Top: Schematic of translation process on localized mRNA-NPs: N-terminal nascent GFP are anchored to mRNA-NP via translating mCherry peptides and ribosome. Bottom: Representative time-lapse images of translated GFP and mCherry with epifluorescence microscopy. e , Cy5 labeled mRNA on NPs are colocalizing with translated GFP. f , Time-dependent evolution of the normalized mean fluorescent intensity of GFP and mCherry in selected regions of interested of the droplets (one frame every 5 minutes). Error bars represent standard deviation (SD). g,h, Representative time-lapse images of the translated GFP from localized mRNA-NPs for mRNA(GFP-122a.a) and mRNA(GFP-375a.a.-mCherry), respectively. i, Time dependent evolution of the mean fluorescent intensity of accumulated GFP on mRNA-NPs for mRNA(GFP-375a.a.-mCherry), mRNA(GFP-122a.a) and mRNA(GFP-mCherry).

Article Snippet: For transcription of Cy5-labeled mRNA 7.5mM CTP was substituted with the mixture of 3.75 mM CTP and 0.25 mM 5-Propargylamino-CTP-CY5 (Jena Bioscience GmbH).

Techniques: Incubation, Epifluorescence Microscopy, Labeling, Concentration Assay, Standard Deviation

a, To assessing the effect of the spatial retention of N-terminal Actin Binding Protein (ABD) during local translation on controlling actin filament assembly, two mRNA sequences were designed by swapping the order of ABD and GFP: ABD-GFP and GFP-ABD. Then mRNAs were translated from localized mRNA-NPs in HeLa extract droplets. The translation of mRNA(GFP-ABD)-NPs were designed to spatially retain N-terminal GFP on the localized mRNA-NPs, whereas the translation of mRNA(ABD-GFP)-NPs will spatially retain N-terminal ABD on the localized mRNA-NPs. b, c, Representative confocal fluorescence microscopy images of Cy5-mRNA-NPs, Phalloidin-568 (F-actin marker), and GFP-ABD ( b ) or ABD-GFP ( c ), after 3 hours of translation. b, zoom: formation of a homogenous F-actin meshwork without any local enrichment of filaments at the vicinity of the mRNA-NPs. c, zoom: formation of a local and dense F-actin meshwork colocalizing with mRNA-NPs. d. Left panel: Representative time-lapse images of F-actin formation upon translation of localized mRNA(ABD-GFP)-NPs. Phalloidin-568 (F-actin marker), and ABD-GFP. Right panel: Quantification of the mean fluorescent intensity of Phalloidin-568 and GFP signal as a function of time in mRNA-NP area. Scale bar, 20 µm ( b-d ).

Journal: bioRxiv

Article Title: Nanoparticle-based local translation reveals mRNA as translation-coupled scaffold with anchoring function

doi: 10.1101/483727

Figure Lengend Snippet: a, To assessing the effect of the spatial retention of N-terminal Actin Binding Protein (ABD) during local translation on controlling actin filament assembly, two mRNA sequences were designed by swapping the order of ABD and GFP: ABD-GFP and GFP-ABD. Then mRNAs were translated from localized mRNA-NPs in HeLa extract droplets. The translation of mRNA(GFP-ABD)-NPs were designed to spatially retain N-terminal GFP on the localized mRNA-NPs, whereas the translation of mRNA(ABD-GFP)-NPs will spatially retain N-terminal ABD on the localized mRNA-NPs. b, c, Representative confocal fluorescence microscopy images of Cy5-mRNA-NPs, Phalloidin-568 (F-actin marker), and GFP-ABD ( b ) or ABD-GFP ( c ), after 3 hours of translation. b, zoom: formation of a homogenous F-actin meshwork without any local enrichment of filaments at the vicinity of the mRNA-NPs. c, zoom: formation of a local and dense F-actin meshwork colocalizing with mRNA-NPs. d. Left panel: Representative time-lapse images of F-actin formation upon translation of localized mRNA(ABD-GFP)-NPs. Phalloidin-568 (F-actin marker), and ABD-GFP. Right panel: Quantification of the mean fluorescent intensity of Phalloidin-568 and GFP signal as a function of time in mRNA-NP area. Scale bar, 20 µm ( b-d ).

Article Snippet: For transcription of Cy5-labeled mRNA 7.5mM CTP was substituted with the mixture of 3.75 mM CTP and 0.25 mM 5-Propargylamino-CTP-CY5 (Jena Bioscience GmbH).

Techniques: Binding Assay, Fluorescence, Microscopy, Marker

Confocal observations of translation from mRNA(GFP-ABD)-NPs ( a ) and mRNA(ABD-GFP)-NPs ( b ) to assess how the spatial organization of mRNA-NP complexes can be regulated by their own protein products. Observations were performed after 2-hour translation. F-actin meshwork is labelled by GFP-ABD and mRNA-NPs by Cy5 labelled-mRNAs. c,d, Time-lapse observations of the dynamic of mRNA translation and mRNA-NP complexes c, N-terminal GFP signals (green) are localized on mRNA(GFP-ABD)-NPs (Red, Cy5 labelled-mRNAs) after 45 minutes of translation. mRNA-NPs are spatially distributed in the droplet and GFP-ABD bound actin filaments form a homogenous meshwork. d, Translation of mRNA(ABD-GFP)-NPs lead to a spatial reorganization of mRNA-NPs as function of time: mRNA(ABD-GFP)-NPs (Red) and ABD-GFP (green) bound to actin filaments spontaneously accumulate together to form a dynamic meshwork that is remodeling spatially. Scale bar, 20 µm.

Journal: bioRxiv

Article Title: Nanoparticle-based local translation reveals mRNA as translation-coupled scaffold with anchoring function

doi: 10.1101/483727

Figure Lengend Snippet: Confocal observations of translation from mRNA(GFP-ABD)-NPs ( a ) and mRNA(ABD-GFP)-NPs ( b ) to assess how the spatial organization of mRNA-NP complexes can be regulated by their own protein products. Observations were performed after 2-hour translation. F-actin meshwork is labelled by GFP-ABD and mRNA-NPs by Cy5 labelled-mRNAs. c,d, Time-lapse observations of the dynamic of mRNA translation and mRNA-NP complexes c, N-terminal GFP signals (green) are localized on mRNA(GFP-ABD)-NPs (Red, Cy5 labelled-mRNAs) after 45 minutes of translation. mRNA-NPs are spatially distributed in the droplet and GFP-ABD bound actin filaments form a homogenous meshwork. d, Translation of mRNA(ABD-GFP)-NPs lead to a spatial reorganization of mRNA-NPs as function of time: mRNA(ABD-GFP)-NPs (Red) and ABD-GFP (green) bound to actin filaments spontaneously accumulate together to form a dynamic meshwork that is remodeling spatially. Scale bar, 20 µm.

Article Snippet: For transcription of Cy5-labeled mRNA 7.5mM CTP was substituted with the mixture of 3.75 mM CTP and 0.25 mM 5-Propargylamino-CTP-CY5 (Jena Bioscience GmbH).

Techniques: