atto488 nhs ester  (Jena Bioscience)

Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A ) Pyrene-actin polymerization assay in the absence or presence of profilin, CapZ, and/or zyxin-VASP or vinculin-VASP condensates. Error bars represent the standard deviations of three independent experiments. ( B ) Schematic of the experimental setup for condensate-mediated actin polymerization on SLBs. ( C and D ) Representative images showing G-actin partitioning into zyxin-VASP (C) and vinculin-VASP (D) condensates. Scale bars, 10 μm. ( E ) Time-lapse image series showing actin polymerization by zyxin-VASP and vinculin-VASP condensates. Scale bars, 1 μm. ( F ) Representative images of actin filaments nucleating outside condensates; arrows indicate filament origin sites, and circles mark condensate positions. Scale bars, 1 μm. ( G ) Box plot quantifying the fraction of actin filaments originating from condensates compared to those originating outside condensates. Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range; P < 0.05 is reported. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Zyxin is visualized with zyxin-mCherry, and actin is visualized with 12.5% actin-ATTO488. Condensates are imaged by epifluorescence microscopy and actin filaments by TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Polymerization Assay, MANN-WHITNEY, Epifluorescence Microscopy, Microscopy

Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A ) Representative image of an actin network generated by zyxin-VASP condensates after 20 min, showing prominent actin bundles. Scale bar, 10 μm. ( B ) Representative image of an actin network generated by vinculin-VASP condensates after 20 min, showing predominantly single filaments. Scale bar, 10 μm. ( C ) Time-lapse image series showing zyxin-VASP–generated filaments forming a bundle upon filament-filament encounter; arrows mark the interaction point. Scale bar, 1 μm. ( D ) Time-lapse image series showing vinculin-VASP–generated filaments crossing over each other upon encounter; arrows mark the interaction point. Scale bar, 1 μm. ( E ) Box plot quantifying actin bundle line density (number of bundles intersecting a line). Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range; P < 0.05 is reported. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Actin is visualized with actin-ATTO488. Actin images are imaged using TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Generated, MANN-WHITNEY, Microscopy

Journal: Science Advances

Article Title: Reconstituted nascent adhesion condensates drive actin polymerization on supported lipid bilayers

doi: 10.1126/sciadv.aeb6691

Figure Lengend Snippet: ( A and B ) Time-lapse image series illustrating changes in condensate shape upon actin polymerization. Scale bars, 5 μm. ( C and D ) Histograms showing changes in condensate eccentricity as a function of actin polymerization. ( E ) Representative FRAP image series of zyxin-VASP and vinculin-VASP condensates. Scale bars, 1 μm. ( F ) Normalized FRAP recovery curves and extracted parameters [fast half-time of recovery ( t 1 / 2 _ fast ) and mobile fraction] for zyxin-VASP and vinculin-VASP condensates. Data are shown as the means ± standard deviation. Error bars represent the standard deviations of three independent experiments. ( G ) Representative image of an actin network generated by zyxin-VASP and subsequently contracted by NMMII in the presence of α-actinin after 60 min, showing clustered actin filaments and condensates. Scale bar, 10 μm. ( H ) Time-lapse image series showing actin network contraction and concomitant clustering of zyxin-VASP condensates in the presence of NMMII and α-actinin. Scale bar, 5 μm. ( I ) Box plot quantifying the number of zyxin-VASP condensates before and after actin network contraction in the presence of NMMII and α-actinin. Boxes indicate the 25th to 75th percentiles, the median is shown as a line, and whiskers represent 1.5× the interquartile range. Significance was tested with a Mann-Whitney U test. Each condition contains n measurements from three independent experiments. Zyxin is visualized with zyxin-mCherry, vinculin is visualized with 10% vinculin-AF555, and actin is visualized with actin-ATTO488. Condensates are imaged using epifluorescence microscopy, and actin filaments are imaged using TIRF microscopy.

Article Snippet: ATTO488 NHS-ester (FP201-488) was purchased from Jena Bioscience.

Techniques: Standard Deviation, Generated, MANN-WHITNEY, Epifluorescence Microscopy, Microscopy

Journal: bioRxiv

Article Title: Plant Root Expansion Microscopy (ROOT-ExM): A streamlined super resolution method for plants

doi: 10.1101/2024.02.21.581184

Figure Lengend Snippet: (A) NHS-ester ATTO488 and NHS-ester ATTO647N labelling. Middle, Imaris 3D reconstruction and segmentation of NHS-ester ATTO488 labelled nuclei and nucleoles. (B-C) STED imaging applied on ROOT-ExM sample labelled with NHS-ester ATTO647. (B) 2D STED. Small inserts show two different compartments identified as mitochondria and plasmodesmata. Intensity profile graphs from the scan lines of the inserts show the resolution of the method. The distance between the mitochondria cristae are approximatively 100 nm while plasmodesmata tube thickness are 27 and 32 nm, very close to their reported diameter in electron microscopy. (C) 3D STED imaging of two plasmodesmata at cell-cell interface from B2. Imaris 3D segmentation of plasmodesmata and orthogonal view showing the isotropic resolution achieved by 3D STED of the plasmodesma channel. ( D ) NHS-ester labelling can be combined with immunolabeling or staining to identify specific structures. (left), NHS-ester ATTO647N labelling combined with KNOLLE (magenta) immunolabeling. (right), NHS-ester ATTO647 labelling combined with calcofluor white staining. Please note that plasmodesmata labelled with NHS-ester ATTO647 are situated in regions characterized by low cellulose content (indicated by arrows and a gap in calcofluor white staining). Scale bar sizes in ROOT-ExM images are calculated by applying the corresponding expansion factor.

Article Snippet: The gels were incubated 1.5h at RT under gentle shaking with NHS-ester ATTO488 (Sigma, 41698), NHS-ester ATTO647 (Sigma, 07376) or NHS-ester ATTO647N (Sigma, 18373) diluted at 20 μg/mL in 100 mM sodium bicarbonate at pH=8.0, then washed 3 times with PBS and expanded.

Techniques: Imaging, Electron Microscopy, Immunolabeling, Staining

Journal: Nature Communications

Article Title: Antigen footprint governs activation of the B cell receptor

doi: 10.1038/s41467-023-36672-0

Figure Lengend Snippet: A A representative RP-HPLC chromatogram of Q1 oligo-NP conjugate purification. B Schematic representation of the core HJ structure and HJs bearing 1–3 NP or NIP haptens. C Native PAGE gel showing the assembly of HJs carrying up to three NP/NIP conjugates and a Q2 oligo bearing AF647 for detection. Green signal is SYBR Gold stain and red signal is AF647. Size marker used is an ultra-low range DNA ladder. D Apparent hydrodynamic radii (R h ) of Atto488-modified HJs with 0–3 units of NP or NIP (50 nM) in Hanks Balanced Salt Solution (HBSS) supplemented with 0.1% BSA (purple) or 5% FCS (green), determined by FIDA. Bars indicate mean ± SD of n = 3 from three independent experiments. E , G , I Bio-layer interferometry (BLI) sensorgrams indicating the binding profile of HJ-NP constructs (0.31–20 nM) to anti-NP mAb (left panels), and modeled distributions of binding kinetics from BLI binding data for haptenated HJ constructs, derived from EVILFIT analysis (right panels). F , H , J Binding profile of HJ-NIP constructs (0.22–14 nM) (left panels), and modeled distributions of binding kinetics from BLI binding data for haptenated HJ constructs, derived from EVILFIT analysis (right panels). Distributions are presented as contour plots on 2D grids with log10(K D ) and log10(k off ) on the x- and y-axes, respectively, at signal max 0.1. Binding traces and the best fits are shown in Supplementary Fig. . Data in ( E – J ) are representative of at least 3 independent experiments with varying conditions. BSA, bovine serum albumin; FCS, fetal calf serum; NP, 4-hydroxy-3-nitrophenylacetyl; NIP, 4-hydroxy-3-iodo-5-nitrophenylacetyl. Source data are provided as a file.

Article Snippet: Q oligos were conjugated to the following two haptens, 4-hydroxy-3-nitrophenylacetyl-O-succinimide ester (NP-Osu) and 4-hydroxy-3-iodo-5-nitrophenylacetyl amino caproyl acetyl-O-succinimide ester (NIP-eCAP-Osu) (Santa Cruz Biotechnology), Alexa Fluor 647 (AF647) or Atto488 NHS ester (Thermo Scientific).

Techniques: Purification, Clear Native PAGE, Staining, Marker, Modification, Binding Assay, Construct, Derivative Assay