snap surface alexa fluor 647  (New England Biolabs)


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    Name:
    SNAP Surface Alexa Fluor 647
    Description:

    Catalog Number:
    S9136
    Price:
    392
    Category:
    Biochemicals
    Applications:
    Cellular Biology
    Size:
    50 nmol
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    Structured Review

    New England Biolabs snap surface alexa fluor 647
    SNAP Surface Alexa Fluor 647

    https://www.bioz.com/result/snap surface alexa fluor 647/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap surface alexa fluor 647 - by Bioz Stars, 2021-07
    96/100 stars

    Images

    1) Product Images from "Correlative Light- and Electron Microscopy with chemical tags"

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2014.03.018

    Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence
    Figure Legend Snippet: Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Techniques Used: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Labeling

    ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm
    Figure Legend Snippet: ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Techniques Used: IA, Fluorescence, Stable Transfection, Expressing, Labeling

    2) Product Images from "Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones"

    Article Title: Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones

    Journal: Science Advances

    doi: 10.1126/sciadv.aay7193

    Analysis of mGluR4 stoichiometry by single-molecule microscopy. ( A ) Schematic view of the mGluR4 construct carrying an N-terminal SNAP-tag (SNAP-mGluR4), which was used for the analysis. The construct was transiently expressed in CHO cells at low densities, corresponding to 0.45 ± 0.08 (SD) fluorescently labeled mGluR4s/μm 2 , and labeled at 1:1 stoichiometry with a saturating concentration of an Alexa Fluor 647 benzylguanine derivative, which binds covalently and irreversibly to the SNAP-tag. Cells were sequentially fixed and imaged by TIRF microscopy. ( B ) Representative TIRF image of a fixed CHO cell expressing the SNAP-mGluR4 construct. Dots represent individual receptor particles, which were identified with an automated single-particle detection algorithm. ( C ) Representative distribution of the intensity of mGluR4 particles in a cell expressing the SNAP-mGluR4 construct. Data were fitted with a mixed Gaussian model. The result of a mixed Gaussian fitting after partial photobleaching (dotted black line) was used to precisely estimate the intensity of single fluorophores in each image sequence. a.u., arbitrary units. ( D ) Relative abundance of monomers, dimers, and higher-order oligomers or nanoclusters detected by the analysis. Data are means ± SEM of 11 cells from three independent experiments (12,012 particles). ( E ) Estimation of the number of primary antibodies binding to one mGluR4. CHO cells transiently transfected to express wild-type mGluR4 at low densities—0.55 ± 0.07 (SD) fluorescently labeled mGluR4s/μm 2 —were incubated with either a limiting dilution (1:10 6 ) or a saturating concentration (1:100) of the primary antibody against mGluR4 and labeled with an Alexa Fluor 647–conjugated secondary antibody. Cells were then imaged and analyzed as in (B) and (C). Representative images and results of 20 (17,257) and 22 (13,553) cells from three independent experiments, respectively (number of particles in brackets), are shown.
    Figure Legend Snippet: Analysis of mGluR4 stoichiometry by single-molecule microscopy. ( A ) Schematic view of the mGluR4 construct carrying an N-terminal SNAP-tag (SNAP-mGluR4), which was used for the analysis. The construct was transiently expressed in CHO cells at low densities, corresponding to 0.45 ± 0.08 (SD) fluorescently labeled mGluR4s/μm 2 , and labeled at 1:1 stoichiometry with a saturating concentration of an Alexa Fluor 647 benzylguanine derivative, which binds covalently and irreversibly to the SNAP-tag. Cells were sequentially fixed and imaged by TIRF microscopy. ( B ) Representative TIRF image of a fixed CHO cell expressing the SNAP-mGluR4 construct. Dots represent individual receptor particles, which were identified with an automated single-particle detection algorithm. ( C ) Representative distribution of the intensity of mGluR4 particles in a cell expressing the SNAP-mGluR4 construct. Data were fitted with a mixed Gaussian model. The result of a mixed Gaussian fitting after partial photobleaching (dotted black line) was used to precisely estimate the intensity of single fluorophores in each image sequence. a.u., arbitrary units. ( D ) Relative abundance of monomers, dimers, and higher-order oligomers or nanoclusters detected by the analysis. Data are means ± SEM of 11 cells from three independent experiments (12,012 particles). ( E ) Estimation of the number of primary antibodies binding to one mGluR4. CHO cells transiently transfected to express wild-type mGluR4 at low densities—0.55 ± 0.07 (SD) fluorescently labeled mGluR4s/μm 2 —were incubated with either a limiting dilution (1:10 6 ) or a saturating concentration (1:100) of the primary antibody against mGluR4 and labeled with an Alexa Fluor 647–conjugated secondary antibody. Cells were then imaged and analyzed as in (B) and (C). Representative images and results of 20 (17,257) and 22 (13,553) cells from three independent experiments, respectively (number of particles in brackets), are shown.

    Techniques Used: Microscopy, Construct, Labeling, Concentration Assay, Expressing, Sequencing, Binding Assay, Transfection, Incubation

    3) Product Images from "Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen"

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0249-x

    Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein
    Figure Legend Snippet: Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Techniques Used: Labeling, SDS Page, Fluorescence, In Vivo Imaging, Staining, SDS-Gel, Marker

    Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )
    Figure Legend Snippet: Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Techniques Used: Binding Assay, Recombinant, Staining, Fluorescence

    4) Product Images from "Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis"

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    Journal: bioRxiv

    doi: 10.1101/2020.05.23.112607

    Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.
    Figure Legend Snippet: Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Techniques Used: Transfection, Construct, Staining

    TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.
    Figure Legend Snippet: TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Techniques Used:

    5) Product Images from "Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding"

    Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding

    Journal: Structure (London, England : 1993)

    doi: 10.1016/j.str.2020.03.001

    Single molecule analysis of FcγR-mAb interactions by fluorescence correlation spectroscopy. Fraction of FcγRs bound to monomeric mAbs alone or previously engaged with their specific antigens. FcγRs were labeled with Alexa-Fluor 647 and their coefficient of diffusion were first determined. Then the receptors were incubated with mAbs wt or LALA (respectively C11 Panels A-B, N5-i5 Panels C-D) alone (Blue bars with a diffusion coefficient of 45 um 2 /sec) or previously engaged in the binding to their specific antigens (FLSC) (Red bars with a diffusion coefficient of 34 um 2 .
    Figure Legend Snippet: Single molecule analysis of FcγR-mAb interactions by fluorescence correlation spectroscopy. Fraction of FcγRs bound to monomeric mAbs alone or previously engaged with their specific antigens. FcγRs were labeled with Alexa-Fluor 647 and their coefficient of diffusion were first determined. Then the receptors were incubated with mAbs wt or LALA (respectively C11 Panels A-B, N5-i5 Panels C-D) alone (Blue bars with a diffusion coefficient of 45 um 2 /sec) or previously engaged in the binding to their specific antigens (FLSC) (Red bars with a diffusion coefficient of 34 um 2 .

    Techniques Used: Fluorescence, Spectroscopy, Labeling, Diffusion-based Assay, Incubation, Binding Assay

    6) Product Images from "Exclusive formation of monovalent quantum dot imaging probes by steric exclusion"

    Article Title: Exclusive formation of monovalent quantum dot imaging probes by steric exclusion

    Journal: Nature methods

    doi: 10.1038/nmeth.2682

    Diffusion dynamics of SNAP-Notch proteins on live cell surfaces ( a ) Cocultures of U2OS cells expressing either SNAP-Notch or Notch-GFP incubated with 1 μM BG-AF647 or 1 μM BG-DNA and complementary mQDs. In both cases, specific labeling of SNAP-Notch proteins was clearly seen by confocal fluorescence microscopy. Scale bar = 10 μm. ( b ) Snapshots from the same region on the same cell showing trajectories of single SNAP-Notch proteins visualized by BG-AF647 and BG-mQD. Scale bar =1 μm. The complete trajectories are shown at the right panel. Some mQDs diffuse in and out of the field of view. ( c ) The mean diffusion constant of at least 15 SNAP-Notch proteins per cell measured with both BG-mQDs or BG-Alexafluor dyes. No statistically significant difference in diffusion was found via t-Test ( p = 0.726). The mean diffusion constant of a SNAP protein fused to an unrelated type I transmembrane domain from CD86 is shown as reference.
    Figure Legend Snippet: Diffusion dynamics of SNAP-Notch proteins on live cell surfaces ( a ) Cocultures of U2OS cells expressing either SNAP-Notch or Notch-GFP incubated with 1 μM BG-AF647 or 1 μM BG-DNA and complementary mQDs. In both cases, specific labeling of SNAP-Notch proteins was clearly seen by confocal fluorescence microscopy. Scale bar = 10 μm. ( b ) Snapshots from the same region on the same cell showing trajectories of single SNAP-Notch proteins visualized by BG-AF647 and BG-mQD. Scale bar =1 μm. The complete trajectories are shown at the right panel. Some mQDs diffuse in and out of the field of view. ( c ) The mean diffusion constant of at least 15 SNAP-Notch proteins per cell measured with both BG-mQDs or BG-Alexafluor dyes. No statistically significant difference in diffusion was found via t-Test ( p = 0.726). The mean diffusion constant of a SNAP protein fused to an unrelated type I transmembrane domain from CD86 is shown as reference.

    Techniques Used: Diffusion-based Assay, Expressing, Incubation, Labeling, Fluorescence, Microscopy

    7) Product Images from "A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies"

    Article Title: A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies

    Journal: Journal of Immunological Methods

    doi: 10.1016/j.jim.2016.03.002

    ADCC assay using cells infected with HIV-1 BaL IMC. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were spinoculated with HIV-1 Bal molecular clone at 2000 rpm for 2 h at 12 °C. After 5 days of co-culture with the virus, cells were washed twice, labeled with Alexa Fluor 647-SNAP tag dye and incubated with dilutions of mAbs (C11, N5-i5, N10-U1 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37 °C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC data represent the typical results obtained in three independent experiments. Upper right panel. The efficiency of the infection was evaluated by staining of the cells with live/dead (not shown), CD4-APC and p24-PE. Lower right panel. Binding of infected EGFP-CEM-NKr-CCR5-SNAP cells with 5 μg/ml Alexa Fluor-647-conjugated mAbs C11 (panel A), N5-i5 (panel B) or N10U1 (panel C).
    Figure Legend Snippet: ADCC assay using cells infected with HIV-1 BaL IMC. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were spinoculated with HIV-1 Bal molecular clone at 2000 rpm for 2 h at 12 °C. After 5 days of co-culture with the virus, cells were washed twice, labeled with Alexa Fluor 647-SNAP tag dye and incubated with dilutions of mAbs (C11, N5-i5, N10-U1 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37 °C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC data represent the typical results obtained in three independent experiments. Upper right panel. The efficiency of the infection was evaluated by staining of the cells with live/dead (not shown), CD4-APC and p24-PE. Lower right panel. Binding of infected EGFP-CEM-NKr-CCR5-SNAP cells with 5 μg/ml Alexa Fluor-647-conjugated mAbs C11 (panel A), N5-i5 (panel B) or N10U1 (panel C).

    Techniques Used: ADCC Assay, Infection, Co-Culture Assay, Labeling, Incubation, Flow Cytometry, Cytometry, Staining, Binding Assay

    Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were generated by stable transfection of EGFP-CEM-NKr cells with Tag-lite pSNAP-CCR5 vector (pSNAP-CCR5, htfr). A–C. GFP and CCR5 expression in parental CEM-NKr-CCR5, EGFP-CEM-NKr and transfected EGFP-CEM-NKr-CCR5-SNAP cells. D–F. Labeling with surface SNAP tag-Alexa Fluor-647 dye vs. GFP expression in the three cell lines. Right panel. The levels of CCR5 and CD4 molecules expressed on EGFP-CEM-NKr-CCR5-SNAP cells in comparison to CEM NKr CCR5 were assessed by flow cytometry with Quantibrite PE calibration beads.
    Figure Legend Snippet: Generation and characterization of EGFP-CEM-NKr-CCR5-SNAP cells. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were generated by stable transfection of EGFP-CEM-NKr cells with Tag-lite pSNAP-CCR5 vector (pSNAP-CCR5, htfr). A–C. GFP and CCR5 expression in parental CEM-NKr-CCR5, EGFP-CEM-NKr and transfected EGFP-CEM-NKr-CCR5-SNAP cells. D–F. Labeling with surface SNAP tag-Alexa Fluor-647 dye vs. GFP expression in the three cell lines. Right panel. The levels of CCR5 and CD4 molecules expressed on EGFP-CEM-NKr-CCR5-SNAP cells in comparison to CEM NKr CCR5 were assessed by flow cytometry with Quantibrite PE calibration beads.

    Techniques Used: Generated, Stable Transfection, Plasmid Preparation, Expressing, Transfection, Labeling, Flow Cytometry, Cytometry

    ADCC assay and mAbs surface staining conducted with cells coated with HIV-1 Bal gp120. A. Comparison of RFADCC assay layout with CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells. CEM NKr CCR5 were stained with PKH-26 and CSFE ( Gomez-Roman et al., 2006 ), while EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye. Killing by C11 mAb (1 μg/ml) is determined as loss of CSFE in PKH26-positive CEM NKr CCR5 cells or loss of GFP in SNAP-positive EGFP-CEM-NKr-CCR5-SNAP cells. B. Cytotoxicity curves for gp120-coated EGFP-CEM-NKr-CCR5-SNAP. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. The binding of 1 μg/ml Alexa-Fluor 647-conjugated mAbs C11 (C and F), N5-i5 (D and G) or N12-i2 (E and H) was compared in gp120-coated-CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells.
    Figure Legend Snippet: ADCC assay and mAbs surface staining conducted with cells coated with HIV-1 Bal gp120. A. Comparison of RFADCC assay layout with CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells. CEM NKr CCR5 were stained with PKH-26 and CSFE ( Gomez-Roman et al., 2006 ), while EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye. Killing by C11 mAb (1 μg/ml) is determined as loss of CSFE in PKH26-positive CEM NKr CCR5 cells or loss of GFP in SNAP-positive EGFP-CEM-NKr-CCR5-SNAP cells. B. Cytotoxicity curves for gp120-coated EGFP-CEM-NKr-CCR5-SNAP. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. The binding of 1 μg/ml Alexa-Fluor 647-conjugated mAbs C11 (C and F), N5-i5 (D and G) or N12-i2 (E and H) was compared in gp120-coated-CEM NKr CCR5 vs. EGFP-CEM-NKr-CCR5-SNAP cells.

    Techniques Used: ADCC Assay, Staining, Binding Assay

    Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells. The optimized assay was modified from Gomez-Roman et al. (2006) . A. For gp120-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP cells were stained with SNAP-Surface Alexa Fluor 647 for 20 min at 37 °C with or without monomeric HIV-1 gp120. B. For AT-2 inactivated virus-based ADCC assay EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye first and then spinoculated with the inactivated virus. C. For IMC-infected targets-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP were spinoculated with IMC, cultured for 5 days, washed twice and then stained with Alexa Fluor 647-SNAP tag dye. Sensitized cells were incubated with dilutions of antibodies for 15 min at room temperature (RT) and subsequently with PBMC as effector cells for 2 or 3 h at 37 °C. Cells were then washed and fixed in 1% paraformaldehyde. The readout is the loss of GFP, as a direct measure of the percentage of target cells cytotoxicity mediated by the mAbs. After coating with monomeric HIV-1 Bal gp120 and adding PBMC as effector cells, we measured the ADCC activity of a reference HIV-1 mAb, C11. The cytotoxicity readout in the original RFADCC was measured as loss of CSFE, while in the modified RFADCC it was the loss of GFP.
    Figure Legend Snippet: Schematic representation of the new RFADCC assay outline conducted with EGFP-CEM-NKr-CCR5-SNAP cells. The optimized assay was modified from Gomez-Roman et al. (2006) . A. For gp120-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP cells were stained with SNAP-Surface Alexa Fluor 647 for 20 min at 37 °C with or without monomeric HIV-1 gp120. B. For AT-2 inactivated virus-based ADCC assay EGFP-CEM-NKr-CCR5-SNAP cells were stained with Alexa Fluor 647-SNAP tag dye first and then spinoculated with the inactivated virus. C. For IMC-infected targets-based ADCC assay, EGFP-CEM-NKr-CCR5-SNAP were spinoculated with IMC, cultured for 5 days, washed twice and then stained with Alexa Fluor 647-SNAP tag dye. Sensitized cells were incubated with dilutions of antibodies for 15 min at room temperature (RT) and subsequently with PBMC as effector cells for 2 or 3 h at 37 °C. Cells were then washed and fixed in 1% paraformaldehyde. The readout is the loss of GFP, as a direct measure of the percentage of target cells cytotoxicity mediated by the mAbs. After coating with monomeric HIV-1 Bal gp120 and adding PBMC as effector cells, we measured the ADCC activity of a reference HIV-1 mAb, C11. The cytotoxicity readout in the original RFADCC was measured as loss of CSFE, while in the modified RFADCC it was the loss of GFP.

    Techniques Used: Modification, ADCC Assay, Staining, Infection, Cell Culture, Incubation, Activity Assay

    ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were labeled with Alexa Fluor 647-SNAP tag and then spinoculated with HIV-1 Bal AT-2 virus at 2000 rpm for 2 h at 12 °C. After two washes, cells were incubated with dilutions of mAbs (C11, N5-i5, N12-i2 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37 °C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. Right panel. The efficiency of the spinoculation was evaluated by cell surface staining with 2 μg/ml Alexa Fluor-594-conjugated mAbs C11 (Panel A), N5-i5 (Panel B) or N12-i2 (panel C).
    Figure Legend Snippet: ADCC assay using cells spinoculated with AT-2 inactivated HIV-1 BaL virions. Left panel. EGFP-CEM-NKr-CCR5-SNAP cells were labeled with Alexa Fluor 647-SNAP tag and then spinoculated with HIV-1 Bal AT-2 virus at 2000 rpm for 2 h at 12 °C. After two washes, cells were incubated with dilutions of mAbs (C11, N5-i5, N12-i2 or Synagis) for 15 min at RT, then PBMC were added to the reaction for 3 h at 37 °C. At the end of the incubation, the samples were washed with PBS, fixed with 1% paraformaldehyde and analyzed by flow cytometry. The ADCC results are representative of three independent assays and the bars indicate the range of the values of cytotoxicity of duplicate samples. Right panel. The efficiency of the spinoculation was evaluated by cell surface staining with 2 μg/ml Alexa Fluor-594-conjugated mAbs C11 (Panel A), N5-i5 (Panel B) or N12-i2 (panel C).

    Techniques Used: ADCC Assay, Labeling, Incubation, Flow Cytometry, Cytometry, Staining

    8) Product Images from "Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis"

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    Journal: bioRxiv

    doi: 10.1101/2020.05.23.112607

    Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.
    Figure Legend Snippet: Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Techniques Used: Transfection, Construct, Staining

    TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.
    Figure Legend Snippet: TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Techniques Used:

    9) Product Images from "Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia"

    Article Title: Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia

    Journal: mAbs

    doi: 10.1080/19420862.2015.1007818

    Internalization of scFv-SNAP‑tag fusion proteins. ( A ) Flow cytometry scFv-SNAP internalization assay. The scFv-SNAP‑tag fusion proteins were labeled with Alexa Fluor 647 and incubated with Kasumi‑1 cells; the fluorescence signal
    Figure Legend Snippet: Internalization of scFv-SNAP‑tag fusion proteins. ( A ) Flow cytometry scFv-SNAP internalization assay. The scFv-SNAP‑tag fusion proteins were labeled with Alexa Fluor 647 and incubated with Kasumi‑1 cells; the fluorescence signal

    Techniques Used: Flow Cytometry, Cytometry, Labeling, Incubation, Fluorescence

    10) Product Images from "A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging"

    Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03191-2

    Comparison and characterization of BC2-nanobody (BC2-Nb) formats for wide-field and dSTORM imaging. a Schematic illustration of the BC2-Nb dye-conjugation strategies. Monovalent and bivalent BC2-Nbs were either conjugated with Alexa Fluor 647 (AF647) via N-hydroxysuccinimide (NHS) ester (left panel) or linked to AF647 by enzymatic sortase coupling (right panel). Wide-field imaging of chemically fixed HeLa cells expressing mCherry-vimentin BC2T (mCherry-VIM BC2T ) stained with modified BC2-Nbs. Monovalent versions of the BC2-Nbs (NHS- and sortase-coupled) are depicted on the left panel, corresponding bivBC2-Nbs are displayed on the right side. Stainings with NHS-conjugated nanobodies are shown in two different image contrasts, the upper half in the same brightness and contrast as the sortase-coupled nanobodies; in the lower half with an adjusted contrast. Scale bars, 25 µm. b Representative dSTORM images of chemically fixed HeLa cells expressing vimentin BC2T , stained with the monomeric NHS-conjugated BC2-Nb AF647 (NHS) (left) and the sortase-coupled bivBC2-Nb AF647 (sort) (right). Scale bars, images 5 µm, insets 1 µm. Image reconstruction details are given in Methods section. c Assessment of staining quality in wide-field fluorescence imaging. Labeling of the different nanobody formats was quantified by calculating the ratio of the signal intensity of mCherry-VIM BC2T expressing cells to non-transfected cells (background), (BC2-Nb AF647 (NHS) : n = 115; bivBC2-Nb AF647 (NHS) : n = 134; BC2-Nb AF647 (sort) : n = 150; bivBC2-Nb AF647 (sort) : n = 195) (Methods section, Supplementary Fig. 1 ). d Assessment of bivBC2-Nb AF647 staining of endogenous β-catenin. Bar chart summarizes measured nanobody per µm 2 values for untransfected chemically fixed HeLa cells stained with GFP-Nb AF647 or bivBC2-Nb AF647 in comparison to chemically fixed HeLa cells transiently expressing BC2T LC3B stained with bivBC2-Nb AF647 , errors given as standard deviation (S.D.), N = 3 cells for each condition (Methods section, Supplementary Fig. 2 ). e Quantification of completeness of labeling for the bivBC2-Nb and SNAP-tag labeling systems using FtnA-oligomers of 24 subunits. Bar chart summarizes median values of FtnA-24mer fluorescence intensities as percentage of theoretical maxima (Methods section, Supplementary Fig. 3 )
    Figure Legend Snippet: Comparison and characterization of BC2-nanobody (BC2-Nb) formats for wide-field and dSTORM imaging. a Schematic illustration of the BC2-Nb dye-conjugation strategies. Monovalent and bivalent BC2-Nbs were either conjugated with Alexa Fluor 647 (AF647) via N-hydroxysuccinimide (NHS) ester (left panel) or linked to AF647 by enzymatic sortase coupling (right panel). Wide-field imaging of chemically fixed HeLa cells expressing mCherry-vimentin BC2T (mCherry-VIM BC2T ) stained with modified BC2-Nbs. Monovalent versions of the BC2-Nbs (NHS- and sortase-coupled) are depicted on the left panel, corresponding bivBC2-Nbs are displayed on the right side. Stainings with NHS-conjugated nanobodies are shown in two different image contrasts, the upper half in the same brightness and contrast as the sortase-coupled nanobodies; in the lower half with an adjusted contrast. Scale bars, 25 µm. b Representative dSTORM images of chemically fixed HeLa cells expressing vimentin BC2T , stained with the monomeric NHS-conjugated BC2-Nb AF647 (NHS) (left) and the sortase-coupled bivBC2-Nb AF647 (sort) (right). Scale bars, images 5 µm, insets 1 µm. Image reconstruction details are given in Methods section. c Assessment of staining quality in wide-field fluorescence imaging. Labeling of the different nanobody formats was quantified by calculating the ratio of the signal intensity of mCherry-VIM BC2T expressing cells to non-transfected cells (background), (BC2-Nb AF647 (NHS) : n = 115; bivBC2-Nb AF647 (NHS) : n = 134; BC2-Nb AF647 (sort) : n = 150; bivBC2-Nb AF647 (sort) : n = 195) (Methods section, Supplementary Fig. 1 ). d Assessment of bivBC2-Nb AF647 staining of endogenous β-catenin. Bar chart summarizes measured nanobody per µm 2 values for untransfected chemically fixed HeLa cells stained with GFP-Nb AF647 or bivBC2-Nb AF647 in comparison to chemically fixed HeLa cells transiently expressing BC2T LC3B stained with bivBC2-Nb AF647 , errors given as standard deviation (S.D.), N = 3 cells for each condition (Methods section, Supplementary Fig. 2 ). e Quantification of completeness of labeling for the bivBC2-Nb and SNAP-tag labeling systems using FtnA-oligomers of 24 subunits. Bar chart summarizes median values of FtnA-24mer fluorescence intensities as percentage of theoretical maxima (Methods section, Supplementary Fig. 3 )

    Techniques Used: Imaging, Conjugation Assay, Expressing, Staining, Modification, Fluorescence, Labeling, Transfection, Standard Deviation

    11) Product Images from "Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro"

    Article Title: Myosin Va’s adaptor protein melanophilin enforces track selection on the microtubule and actin networks in vitro

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1619473114

    Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)
    Figure Legend Snippet: Mlph’s phosphorylation state does not interfere substantially with actin binding. ( A ) Actin decoration experiments were performed with surface-immobilized and Atto488-labeled actin filaments (red). Filaments were incubated with the complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). Dephosphorylated (Dephos; Left ) and the phosphorylated (Phos; Right ) Mlph decorated actin filaments similarly well. Removal of the C-terminal ABD of Mlph (Rab27a/Mlph ΔABD) abolished this interaction regardless of Mlph’s phosphorylation state. ( B ) The dephosphorylated, Alexa Fluor 488-labeled Rab27a/Mlph complex was mixed in equal amounts with the phosphorylated, Alexa Fluor 647-labeled Rab27a/Mlph complex and was incubated with surface-attached, Atto565-labeled actin filaments. The quantification of the actin-associated fluorescence signals from the respective PKA- and phosphatase-treated Rab27a/Mlph complexes showed that the phosphorylation state of Mlph did not substantially interfere with actin binding. Error bars represent SD. (Scale bars: 3 µm.)

    Techniques Used: Binding Assay, Labeling, Incubation, Fluorescence

    Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Surface-immobilized and Atto565-labeled actin filaments (red; Upper ) and Atto488-labeled microtubules (red; Lower ) were incubated with phosphorylated ( A ) and dephosphorylated ( B ) complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). ( A ) The phosphorylated Rab27a/Mlph complex largely ignored microtubules (MT) and associated with actin filaments (17 ± 4% vs. 83 ± 4%). ( B ) Upon dephosphorylation, the behavior of the Rab27a/Mlph complex was reversed, and the microtubule binding clearly dominated (76 ± 4%) over the actin binding (24 ± 4%). Error bars represent SD. (Scale bar: 3 µm.)
    Figure Legend Snippet: Dephosphorylation is sufficient to relocate Mlph from actin to microtubules efficiently. Surface-immobilized and Atto565-labeled actin filaments (red; Upper ) and Atto488-labeled microtubules (red; Lower ) were incubated with phosphorylated ( A ) and dephosphorylated ( B ) complex formed between Mlph and Alexa Fluor 647-labeled Rab27a (green). ( A ) The phosphorylated Rab27a/Mlph complex largely ignored microtubules (MT) and associated with actin filaments (17 ± 4% vs. 83 ± 4%). ( B ) Upon dephosphorylation, the behavior of the Rab27a/Mlph complex was reversed, and the microtubule binding clearly dominated (76 ± 4%) over the actin binding (24 ± 4%). Error bars represent SD. (Scale bar: 3 µm.)

    Techniques Used: De-Phosphorylation Assay, Labeling, Incubation, Binding Assay

    Mlph interacts with microtubules in a phosphorylation-dependent manner. In microtubule decoration experiments Atto488-labeled microtubules (red) were incubated with the Alexa Fluor 647-labeled Rab27a/Mlph complex (green). Decoration of microtubules was strictly dependent on the phosphorylation state of Mlph. The fluorescent background from the phosphorylated and dephosphorylated Rab27a/Mlph complex seen in the green channel was comparable, indicating similar amounts of protein. (Scale bar: 3 µm.)
    Figure Legend Snippet: Mlph interacts with microtubules in a phosphorylation-dependent manner. In microtubule decoration experiments Atto488-labeled microtubules (red) were incubated with the Alexa Fluor 647-labeled Rab27a/Mlph complex (green). Decoration of microtubules was strictly dependent on the phosphorylation state of Mlph. The fluorescent background from the phosphorylated and dephosphorylated Rab27a/Mlph complex seen in the green channel was comparable, indicating similar amounts of protein. (Scale bar: 3 µm.)

    Techniques Used: Labeling, Incubation

    12) Product Images from "Correlative Light- and Electron Microscopy with chemical tags"

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    Journal: Journal of structural biology

    doi: 10.1016/j.jsb.2014.03.018

    Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence
    Figure Legend Snippet: Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Techniques Used: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Labeling

    ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm
    Figure Legend Snippet: ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Techniques Used: IA, Fluorescence, Stable Transfection, Expressing, Labeling

    13) Product Images from "DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes"

    Article Title: DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

    Journal: bioRxiv

    doi: 10.1101/092791

    Effects of DYNC1H1 mutations on the assembly and processivity of dynein-dynactin-BICD2N complexes. (A) Kymographs showing examples of frequent co-localisation of mutant TMR-dynein complexes (green) with Alexa647-BICD2N (magenta) on microtubules in the presence of dynactin (see Supplementary Figures 4 and 5 for examples for other mutant complexes). Co-localisation indicates formation of a dynein-dynactin-BICD2N complex. White, blue and yellow arrowheads indicate examples of co-localisation of Alexa647-BICD2N with TMR-dynein exhibiting processive, static and diffusive behaviour, respectively. Red arrowheads indicate examples of TMR-dynein complexes that do not have an Alexa647-BICD2N signal. (B) Quantification of the percentage of all microtubule-associated dyneins that co-localises with BICD2N in the presence of dynactin. (C–E) Quantification of the percentage of microtubule-associated dynein-dynactin-BICD2N (DDB) complexes that exhibits processive (C), static (D) and diffusive (E) behaviour. In B–E, each magenta circle represents the mean value for an individual chamber (at least 70 dynein complexes (B) and 50 dynein-dynactin-BICD2N (C–E) complexes analysed per chamber). Grey bars show means of the individual chamber values for each condition, with error bars representing S.E.M. Statistical significance, compared to WT, was evaluated with a one-way ANOVA with Sidak’s multiple comparison test (***, p
    Figure Legend Snippet: Effects of DYNC1H1 mutations on the assembly and processivity of dynein-dynactin-BICD2N complexes. (A) Kymographs showing examples of frequent co-localisation of mutant TMR-dynein complexes (green) with Alexa647-BICD2N (magenta) on microtubules in the presence of dynactin (see Supplementary Figures 4 and 5 for examples for other mutant complexes). Co-localisation indicates formation of a dynein-dynactin-BICD2N complex. White, blue and yellow arrowheads indicate examples of co-localisation of Alexa647-BICD2N with TMR-dynein exhibiting processive, static and diffusive behaviour, respectively. Red arrowheads indicate examples of TMR-dynein complexes that do not have an Alexa647-BICD2N signal. (B) Quantification of the percentage of all microtubule-associated dyneins that co-localises with BICD2N in the presence of dynactin. (C–E) Quantification of the percentage of microtubule-associated dynein-dynactin-BICD2N (DDB) complexes that exhibits processive (C), static (D) and diffusive (E) behaviour. In B–E, each magenta circle represents the mean value for an individual chamber (at least 70 dynein complexes (B) and 50 dynein-dynactin-BICD2N (C–E) complexes analysed per chamber). Grey bars show means of the individual chamber values for each condition, with error bars representing S.E.M. Statistical significance, compared to WT, was evaluated with a one-way ANOVA with Sidak’s multiple comparison test (***, p

    Techniques Used: Mutagenesis

    14) Product Images from "Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization"

    Article Title: Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

    Journal: Biomedical Optics Express

    doi: 10.1364/BOE.10.002708

    Imaging of nuclear pore complex protein Nup107 about 5 µm deep in the cell. (a) Nup107-SNAP-Alexa Fluor 647 imaged with dSTORM reconstructed by the PSF model obtained on the coverslip. (b) dSTORM image after applying the depth dependent z correction to a. (c) Side-view reconstruction of the region bounded by dashed line in a. (d) Side-view reconstruction of the same region as in c after applying the depth-dependent z -correction. Scale bars, 1 µm.
    Figure Legend Snippet: Imaging of nuclear pore complex protein Nup107 about 5 µm deep in the cell. (a) Nup107-SNAP-Alexa Fluor 647 imaged with dSTORM reconstructed by the PSF model obtained on the coverslip. (b) dSTORM image after applying the depth dependent z correction to a. (c) Side-view reconstruction of the region bounded by dashed line in a. (d) Side-view reconstruction of the same region as in c after applying the depth-dependent z -correction. Scale bars, 1 µm.

    Techniques Used: Imaging

    Imaging of Nup107 at 340 nm above the coverslip. (a) Nup107-SNAP-Alexa Fluor 647 imaged with dSTORM reconstructed by the PSF model obtained on the coverslip. (b) dSTORM image of a representative single NPC as indicated in the squared region in (a). Top images are x - y and x - z views. Bottom image is the intensity plot along z and a two Gaussian model was fitted on the data. (c) Side-view reconstruction of the region bounded by dashed lines in (a). (d) The distance of the two rings as a function of the central z position of each individual NPC before correction (Pearson correlation coefficient c = 0.55). (e) The distance of the two rings as a function of the central z position of each individual NPC after correction (Pearson correlation coefficient c = −0.03). Scale bars, (a) and (c) 1 µm, (b) 50 nm.
    Figure Legend Snippet: Imaging of Nup107 at 340 nm above the coverslip. (a) Nup107-SNAP-Alexa Fluor 647 imaged with dSTORM reconstructed by the PSF model obtained on the coverslip. (b) dSTORM image of a representative single NPC as indicated in the squared region in (a). Top images are x - y and x - z views. Bottom image is the intensity plot along z and a two Gaussian model was fitted on the data. (c) Side-view reconstruction of the region bounded by dashed lines in (a). (d) The distance of the two rings as a function of the central z position of each individual NPC before correction (Pearson correlation coefficient c = 0.55). (e) The distance of the two rings as a function of the central z position of each individual NPC after correction (Pearson correlation coefficient c = −0.03). Scale bars, (a) and (c) 1 µm, (b) 50 nm.

    Techniques Used: Imaging

    Related Articles

    Purification:

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen
    Article Snippet: The protein concentration was determined using the AIDA Image Analyzer as described above. .. Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ]. .. Briefly, 1 μg SNAP-TTC protein was mixed with 2 nmol BG-647 or BG-488 solution prepared from a 50 nmol stock and incubated for 1 h at room temperature.

    Staining:

    Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding
    Article Snippet: ADCC was assessed using the rapid fluorescence antibody-dependent cellular cytotoxicity assay (RFADCC) ( ) as modified in our laboratory for high-throughput assays ( ). .. Briefly, EGFP-CEM-NKr-CCR5SNAP cells stained with SNAP-Surface Alexa Fluor 647 (New England BioLabs) and sensitized with recombinant HIV-1 Ba-L gp120 were used as targets and PBMC were utilized as effectors. .. All the antibodies activity was analyzed with four-fold serial dilutions starting from a concentration of 0.3 ug/ml.

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: At 24-hour post seeding, the cells were transfected with 100 ng of either SNAP-FL, SNAP-ΔG, or SNAP-ΔGΔH-EAP45 expressor. .. This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day. .. A primary antibody to RAB7 (Abcam) or anti-Tubulin (Sigma) was also added on some occasions followed by staining with the secondary antibody conjugated with either AlexaFluor488 or AlexaFluor647 (Thermofisher) before imaging using Leica SP5 confocal fluorescence microscope.

    Article Title: Correlative Light- and Electron Microscopy with chemical tags
    Article Snippet: L cells stably expressing Ncad-SNAPf were grown to a confluency of 60–80%, stained in 2 μM SNAP-Surface Alexa Fluor 647 solution (dilution in phenolred free complete medium) at 37 °C for 5 min and subsequently washed three times with phenolred free complete medium. .. For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used. .. After 30 min of staining, samples were rinsed three times and washed for at least 30 min with OptiMEM with 10% FBS.

    Article Title: A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies
    Article Snippet: 2.3 Optimized RFADCC assay and cell surface staining To optimize the RFADCC assay ( ) for high throughput efficiency, the regular double staining with the membrane PKH-26 dye and the intracellular carboxyfluorescein diacetate, succinimidyl ester (CSFE) dye were replaced with the membrane staining of the CCR5-SNAP-tag and the constitutive intracellular expression of GFP. .. For the ADCC protocol , EGFP-CEM-NKr-CCR5-SNAP target cells were stained with the fluorescent substrate SNAP-Surface Alexa Fluor 647 (New England BioLabs Cat. S9136S) for 20 min at 37 °C with or without coating of monomeric HIV-1 Bal gp120 (50 μg/ml). .. For the studies with spinoculated virus, the cells were first stained with the SNAP-Surface dye and then spinoculated with the AT-2 inactivated Bal HIV-1 virus at 2000 RPM for 2 h at 12 °C.

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: .. At 24 hours post transfection, the cells were fixed, permeabilised and stained with the SNAP-Surface AlexaFluor647 (NEB) at 4 µM for 1 hour. ..

    Recombinant:

    Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding
    Article Snippet: ADCC was assessed using the rapid fluorescence antibody-dependent cellular cytotoxicity assay (RFADCC) ( ) as modified in our laboratory for high-throughput assays ( ). .. Briefly, EGFP-CEM-NKr-CCR5SNAP cells stained with SNAP-Surface Alexa Fluor 647 (New England BioLabs) and sensitized with recombinant HIV-1 Ba-L gp120 were used as targets and PBMC were utilized as effectors. .. All the antibodies activity was analyzed with four-fold serial dilutions starting from a concentration of 0.3 ug/ml.

    Concentration Assay:

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: At 24-hour post seeding, the cells were transfected with 100 ng of either SNAP-FL, SNAP-ΔG, or SNAP-ΔGΔH-EAP45 expressor. .. This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day. .. A primary antibody to RAB7 (Abcam) or anti-Tubulin (Sigma) was also added on some occasions followed by staining with the secondary antibody conjugated with either AlexaFluor488 or AlexaFluor647 (Thermofisher) before imaging using Leica SP5 confocal fluorescence microscope.

    Incubation:

    Article Title: Exclusive formation of monovalent quantum dot imaging probes by steric exclusion
    Article Snippet: Live cell labeling and imaging Unless otherwise noted, cells were incubated with 1 μM BG-DNA for 30 minutes at 37 °C, washed three times with PBS containing 1 % BSA, and then incubated with 200 pM mQDs for 5 minutes at room temperature before a final wash with 1 % BSA. .. In dye-comparison experiments, cells were incubated simultaneously with 1 μM BG-DNA and 0.2 μM BG-AF647 (Surface-SNAP-647, NEB). .. For single particle tracking, mQDs were incubated in PBS containing 1 % BSA for 30 min at room temperature prior to being added to cells at a concentration of 0.2 nM.

    Transfection:

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis
    Article Snippet: .. At 24 hours post transfection, the cells were fixed, permeabilised and stained with the SNAP-Surface AlexaFluor647 (NEB) at 4 µM for 1 hour. ..

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    New England Biolabs snap surface alexa fluor 647
    Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with <t>Alexa</t> Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence
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    Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Journal: Journal of structural biology

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    doi: 10.1016/j.jsb.2014.03.018

    Figure Lengend Snippet: Live-cell imaging and post-embedding fluorescence at different UA levels. (a, b) Live-cell imaging of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647. The cell–cell junctions are visualized in the red channel. The auto-fluorescence

    Article Snippet: For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used.

    Techniques: Live Cell Imaging, Fluorescence, Stable Transfection, Expressing, Labeling

    ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Journal: Journal of structural biology

    Article Title: Correlative Light- and Electron Microscopy with chemical tags

    doi: 10.1016/j.jsb.2014.03.018

    Figure Lengend Snippet: ia-SEM CLEM of NCadherin in L-cells. (a) Fluorescence image of L-cells stably expressing SNAPf-NCadherin labeled with Alexa Fluor 647 after high-pressure freezing and freeze substitution with 2% UA. The box outlines the 32 μm × 17 μm

    Article Snippet: For staining of HeLa cells, 3 μM SNAP-Cell TMR Star, 2 μM SNAP-Surface Alexa Fluor 647 (New England BioLabs), 3 μM SiR-SNAP ( ) and 50 μM HaloTag TMR Ligand (Promega) diluted in OptiMEM (Life Technologies) with 10% FBS were used.

    Techniques: IA, Fluorescence, Stable Transfection, Expressing, Labeling

    Analysis of mGluR4 stoichiometry by single-molecule microscopy. ( A ) Schematic view of the mGluR4 construct carrying an N-terminal SNAP-tag (SNAP-mGluR4), which was used for the analysis. The construct was transiently expressed in CHO cells at low densities, corresponding to 0.45 ± 0.08 (SD) fluorescently labeled mGluR4s/μm 2 , and labeled at 1:1 stoichiometry with a saturating concentration of an Alexa Fluor 647 benzylguanine derivative, which binds covalently and irreversibly to the SNAP-tag. Cells were sequentially fixed and imaged by TIRF microscopy. ( B ) Representative TIRF image of a fixed CHO cell expressing the SNAP-mGluR4 construct. Dots represent individual receptor particles, which were identified with an automated single-particle detection algorithm. ( C ) Representative distribution of the intensity of mGluR4 particles in a cell expressing the SNAP-mGluR4 construct. Data were fitted with a mixed Gaussian model. The result of a mixed Gaussian fitting after partial photobleaching (dotted black line) was used to precisely estimate the intensity of single fluorophores in each image sequence. a.u., arbitrary units. ( D ) Relative abundance of monomers, dimers, and higher-order oligomers or nanoclusters detected by the analysis. Data are means ± SEM of 11 cells from three independent experiments (12,012 particles). ( E ) Estimation of the number of primary antibodies binding to one mGluR4. CHO cells transiently transfected to express wild-type mGluR4 at low densities—0.55 ± 0.07 (SD) fluorescently labeled mGluR4s/μm 2 —were incubated with either a limiting dilution (1:10 6 ) or a saturating concentration (1:100) of the primary antibody against mGluR4 and labeled with an Alexa Fluor 647–conjugated secondary antibody. Cells were then imaged and analyzed as in (B) and (C). Representative images and results of 20 (17,257) and 22 (13,553) cells from three independent experiments, respectively (number of particles in brackets), are shown.

    Journal: Science Advances

    Article Title: Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones

    doi: 10.1126/sciadv.aay7193

    Figure Lengend Snippet: Analysis of mGluR4 stoichiometry by single-molecule microscopy. ( A ) Schematic view of the mGluR4 construct carrying an N-terminal SNAP-tag (SNAP-mGluR4), which was used for the analysis. The construct was transiently expressed in CHO cells at low densities, corresponding to 0.45 ± 0.08 (SD) fluorescently labeled mGluR4s/μm 2 , and labeled at 1:1 stoichiometry with a saturating concentration of an Alexa Fluor 647 benzylguanine derivative, which binds covalently and irreversibly to the SNAP-tag. Cells were sequentially fixed and imaged by TIRF microscopy. ( B ) Representative TIRF image of a fixed CHO cell expressing the SNAP-mGluR4 construct. Dots represent individual receptor particles, which were identified with an automated single-particle detection algorithm. ( C ) Representative distribution of the intensity of mGluR4 particles in a cell expressing the SNAP-mGluR4 construct. Data were fitted with a mixed Gaussian model. The result of a mixed Gaussian fitting after partial photobleaching (dotted black line) was used to precisely estimate the intensity of single fluorophores in each image sequence. a.u., arbitrary units. ( D ) Relative abundance of monomers, dimers, and higher-order oligomers or nanoclusters detected by the analysis. Data are means ± SEM of 11 cells from three independent experiments (12,012 particles). ( E ) Estimation of the number of primary antibodies binding to one mGluR4. CHO cells transiently transfected to express wild-type mGluR4 at low densities—0.55 ± 0.07 (SD) fluorescently labeled mGluR4s/μm 2 —were incubated with either a limiting dilution (1:10 6 ) or a saturating concentration (1:100) of the primary antibody against mGluR4 and labeled with an Alexa Fluor 647–conjugated secondary antibody. Cells were then imaged and analyzed as in (B) and (C). Representative images and results of 20 (17,257) and 22 (13,553) cells from three independent experiments, respectively (number of particles in brackets), are shown.

    Article Snippet: SNAP-Surface Alexa Fluor 647 was from New England Biolabs (Ipswich, MA, USA).

    Techniques: Microscopy, Construct, Labeling, Concentration Assay, Expressing, Sequencing, Binding Assay, Transfection, Incubation

    Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Fluorescent in-gel detection of SNAP-TTC labeled with different dyes. a SDS-PAGE of SNAP-TTC fusion protein labeled with SNAP-Surface® Alexa Fluor® 488 (2) or BG-647 (3), respectively. Fluorescence signals were visualized using the Maestro CRi in vivo imaging system with the appropriate filter set. b Coomassie-stained SDS gel from ( a ). The stained protein bands correspond to the measured fluorescence signals from ( a ). (1) prestained protein marker, broad range (NEB), (2) SNAP-TTC-SNAP-Surface® Alexa Fluor® 488, (3) SNAP-TTC-BG647, (4) uncoupled SNAP-TTC protein

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Labeling, SDS Page, Fluorescence, In Vivo Imaging, Staining, SDS-Gel, Marker

    Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Journal: BMC Biotechnology

    Article Title: Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen

    doi: 10.1186/s12896-016-0249-x

    Figure Lengend Snippet: Binding analysis of recombinant TTC-based proteins to TTC-reactive hybridoma cells. Equimolar amounts (100 nM) of TTC ( c ) and TTC-ETA’ ( d ) were used for binding analysis to the TTC-reactive hybridoma cell line 5E4 ( a ) compared to the control hybridoma cell line 8.18-C5 ( b ). Detection of bound proteins was carried out using an Alexa Fluor® 488-coupled anti-His5 antibody. Staining with Alexa Fluor 488-coupled anti-His5 antibody ( b ) and unstained cells ( a ) served as controls. Binding analysis of 100 nM SNAP-TTC coupled to the SNAP-Surface® 647 fluorescence dye ( b ) to 5E4 hybridoma cells ( c ) and to the control hybridoma cell line 8.18-C5 ( d ). Unstained cells served as control ( a )

    Article Snippet: Coupling SNAP-TTC to the fluorescent dye Purified SNAP-TTC was conjugated to the BG-modified fluorescent dyes SNAP-surface® Alexa Fluor® 647 (New England Biolabs, Frankfurt am Main, Germany; Catalogue number: S9136S) and SNAP-surface® Alexa Fluor® 488 (New England Biolabs; Catalogue number: S9129S) as previously described [ ].

    Techniques: Binding Assay, Recombinant, Staining, Fluorescence

    Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Journal: bioRxiv

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    doi: 10.1101/2020.05.23.112607

    Figure Lengend Snippet: Glue domain is not required for the localisation of EAP45 at the late endosome, but it is required for its localisation at the intercellular bridge during cytokinesis. Confocal images of HeLa cells (A) and YFP-TSG101 HeLa cells (B) transfected with different SNAP-EAP45 constructs, and stained one day post-transfection with DAPI for nuclei and AlexaFluor647 conjugated SNAP tag substrates for EAP45. The HeLa cells in (A) were stained with RAB7, to visualise the colocalisation of late endosomes and EAP45. Close-up views in yellow insets highlight areas of colocalisation. The YFP-TSG101 HeLa cells (B) show the colocalisation of TSG101 and EAP45 is similar for the FL and ΔG constructs, but reduced upon deletion of the H0 linker region. (C) Confocal images of HeLa cells stained with DAPI, α -tubulin, and AlexaFluor647-SNAP substrates for EAP45. (D) Plots of line density profiles across the intercellular bridge for each condition shown in (C) . (E) Quantification of the intensities for each condition where EAP45 is recruited at the intercellular bridge is shown.

    Article Snippet: This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day.

    Techniques: Transfection, Construct, Staining

    TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Journal: bioRxiv

    Article Title: Distinct domain requirements for EAP45 in HIV budding, late endosomal recruitment, and cytokinesis

    doi: 10.1101/2020.05.23.112607

    Figure Lengend Snippet: TIRF image of GFP-Gag and anti-GFP nanobody conjugated AlexaFluor647 to test the maximal experimentally attainable colocalisation. (A) shows a representative dual-colour image of GFP-Gag and the AlexaFluor647 nanobody, with a faded-in composite towards the lower right corner showing the resulting dual-colour mask after Weka segmentation ( 1 ). Panel (B) (n=5 cells) displays the percentage of Gag particles with at least one AlexaFluor647 particle in proximity within a given radius, as a function of the search radius.

    Article Snippet: This was followed by fixation with PFA and staining with SNAP-Surface AlexaFluor647 (NEB) at 4 µM concentration for SNAP-EAP45 and DAPI for nuclei on the following day.

    Techniques: