snap surface  (New England Biolabs)


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    New England Biolabs snap surface
    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.
    Snap Surface, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    Images

    1) Product Images from "Choosing the Probe for Single-Molecule Fluorescence Microscopy"

    Article Title: Choosing the Probe for Single-Molecule Fluorescence Microscopy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232314949

    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.
    Figure Legend Snippet: Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.

    Techniques Used: Imaging, Labeling, Diffusion-based Assay, Single Particle, Fluorescence, Spectroscopy, Incubation, Transfection

    snap surface  (New England Biolabs)


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    New England Biolabs snap surface
    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.
    Snap Surface, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface/product/New England Biolabs
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    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Choosing the Probe for Single-Molecule Fluorescence Microscopy"

    Article Title: Choosing the Probe for Single-Molecule Fluorescence Microscopy

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232314949

    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.
    Figure Legend Snippet: Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.

    Techniques Used: Imaging, Labeling, Diffusion-based Assay, Single Particle, Fluorescence, Spectroscopy, Incubation, Transfection

    snap substrates  (New England Biolabs)


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    New England Biolabs snap substrates
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion"

    Article Title: Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2022.101541

    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Figure Legend Snippet: Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Techniques Used: Construct, Clone Assay, Western Blot, Incubation, Flow Cytometry, Labeling

    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface block/product/New England Biolabs
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    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS <t>SNAP-smo</t> wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale <t>grey),</t> <t>intracellular</t> (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface block/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
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    Images

    1) Product Images from "High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened"

    Article Title: High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened

    Journal: bioRxiv

    doi: 10.1101/2022.01.19.476759

    (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS SNAP-smo wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale grey), intracellular (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.
    Figure Legend Snippet: (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS SNAP-smo wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale grey), intracellular (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.

    Techniques Used: Expressing, Labeling, Staining, Immunolabeling

    snap surface probes  (New England Biolabs)


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    New England Biolabs snap surface probes
    Snap Surface Probes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface probes  (New England Biolabs)


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    New England Biolabs snap surface probes
    Snap Surface Probes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface block 480  (New England Biolabs)


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    New England Biolabs snap surface block 480
    Snap Surface Block 480, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    CBG <t>and</t> <t>SBG-conjugated</t> SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally <t>SNAP-fused</t> GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates"

    Article Title: Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

    Journal: Chemical Science

    doi: 10.1039/d0sc02794d

    CBG and SBG-conjugated SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.
    Figure Legend Snippet: CBG and SBG-conjugated SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.

    Techniques Used: Concentration Assay

    SBG-conjugated fluorophores across the visible spectrum enable surface-specific SNAP labelling and nanoscopic imaging of surface receptors. (a–d) SBG conjugation enables surface targeting of Oregon Green (a), TMR (b), JF 549 (c), and JF 646 (d). All fluorophores label nuclear SNAP-tags when conjugated BG but not SBG, for which they show cleaner surface targeting of SNAP-mGluR2. (e and f) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 (e) and SBG-JF 646 (f) shows clear isolation of the membrane population only using the impermeable SBG probe. Labelling concentration was 500 nM for all compounds. Data is represented as mean ± SEM. Scale bars are 20 μm.
    Figure Legend Snippet: SBG-conjugated fluorophores across the visible spectrum enable surface-specific SNAP labelling and nanoscopic imaging of surface receptors. (a–d) SBG conjugation enables surface targeting of Oregon Green (a), TMR (b), JF 549 (c), and JF 646 (d). All fluorophores label nuclear SNAP-tags when conjugated BG but not SBG, for which they show cleaner surface targeting of SNAP-mGluR2. (e and f) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 (e) and SBG-JF 646 (f) shows clear isolation of the membrane population only using the impermeable SBG probe. Labelling concentration was 500 nM for all compounds. Data is represented as mean ± SEM. Scale bars are 20 μm.

    Techniques Used: Imaging, Conjugation Assay, Isolation, Concentration Assay

    In vivo labelling of a SNAP-tagged receptor with SBG-conjugated fluorophores produces less background and more spread. (a) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex (mPFC) of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. (b and c) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG (b) or SBG (c) fluorophores. (d) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t -test, p = 0.04). (e and f) Representative images showing fluorescence in control slices following injection of BG (e) or SBG (f) fluorophores. (g) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t -test, p = 0.007). Data is represented as mean ± SEM and comes from n = 3 mice for each condition. Labelling concentration was 1 μM. Scale bars are 150 μm.
    Figure Legend Snippet: In vivo labelling of a SNAP-tagged receptor with SBG-conjugated fluorophores produces less background and more spread. (a) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex (mPFC) of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. (b and c) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG (b) or SBG (c) fluorophores. (d) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t -test, p = 0.04). (e and f) Representative images showing fluorescence in control slices following injection of BG (e) or SBG (f) fluorophores. (g) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t -test, p = 0.007). Data is represented as mean ± SEM and comes from n = 3 mice for each condition. Labelling concentration was 1 μM. Scale bars are 150 μm.

    Techniques Used: In Vivo, Expressing, Injection, Slice Preparation, Fluorescence, Concentration Assay

    BG and SBG-conjugated fluorophores enable SiMPull analysis of isolated surface or intracellular receptor populations. (a) Representative images showing labelling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or SNAP-Surface® Block followed by BG-JF 549 , respectively. (b) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labelling. (c) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. (d) Summary of the proportion of 2-step bleaching steps for each labelling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p = 0.0005). (e–h) Same as (a–d) but with SNAP-β2AR. * indicates statistical significance (unpaired t test, p = 0.008). Scale bars are 10 μm. (i) Representative images showing 2-colour labelling of either surface SNAP-mGluR2 exclusively (top) or surface and intracellular SNAP-mGluR2 (bottom). All fluorophores were applied at 1 μM. (j) Representative images of single molecules in two different colours, with co-localized spots circled. (k) Summary of the proportion of total spots that are co-localized between the two colours. Each point represents one independent movie and bars show mean ± SEM.
    Figure Legend Snippet: BG and SBG-conjugated fluorophores enable SiMPull analysis of isolated surface or intracellular receptor populations. (a) Representative images showing labelling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or SNAP-Surface® Block followed by BG-JF 549 , respectively. (b) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labelling. (c) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. (d) Summary of the proportion of 2-step bleaching steps for each labelling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p = 0.0005). (e–h) Same as (a–d) but with SNAP-β2AR. * indicates statistical significance (unpaired t test, p = 0.008). Scale bars are 10 μm. (i) Representative images showing 2-colour labelling of either surface SNAP-mGluR2 exclusively (top) or surface and intracellular SNAP-mGluR2 (bottom). All fluorophores were applied at 1 μM. (j) Representative images of single molecules in two different colours, with co-localized spots circled. (k) Summary of the proportion of total spots that are co-localized between the two colours. Each point represents one independent movie and bars show mean ± SEM.

    Techniques Used: Isolation, Blocking Assay

    snap surface block  (New England Biolabs)


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    Structured Review

    New England Biolabs snap surface block
    a ) Application of permeable BG-SiR to N-terminally <t>SNAP-fused</t> GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of <t>impermeable</t> <t>SBG-SiR</t> should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates"

    Article Title: Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924829

    a ) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.
    Figure Legend Snippet: a ) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.

    Techniques Used: Concentration Assay, Labeling

    a-d ) SBG conjugation enables surface targeting of Oregon Green ( a ), TMR ( b ), JF 549 ( c ), and JF 646 ( d ). All fluorophores are able to label nuclear SNAP-tags when conjugated BG but not SBG and show cleaner surface targeting of SNAP-mGluR2. e, f ) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 ( e ) and SBG-JF 646 ( f ) shows clear isolation of the membrane population only using the impermeable SBG probe. Data is represented as mean ± SEM. Scale bars are 20 µm.
    Figure Legend Snippet: a-d ) SBG conjugation enables surface targeting of Oregon Green ( a ), TMR ( b ), JF 549 ( c ), and JF 646 ( d ). All fluorophores are able to label nuclear SNAP-tags when conjugated BG but not SBG and show cleaner surface targeting of SNAP-mGluR2. e, f ) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 ( e ) and SBG-JF 646 ( f ) shows clear isolation of the membrane population only using the impermeable SBG probe. Data is represented as mean ± SEM. Scale bars are 20 µm.

    Techniques Used: Conjugation Assay, Isolation

    a ) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. b-c ) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG ( b ) or SBG ( c ) fluorophores. d ) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t-test, p=0.04). e, f ) Representative images showing fluorescence in control slices following injection of BG ( e ) or SBG ( f ) fluorophores. g ) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t-test, p=0.007). Data is represented as mean ± SEM and comes from n=3 mice for each condition. Scale bars are 150 µm.
    Figure Legend Snippet: a ) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. b-c ) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG ( b ) or SBG ( c ) fluorophores. d ) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t-test, p=0.04). e, f ) Representative images showing fluorescence in control slices following injection of BG ( e ) or SBG ( f ) fluorophores. g ) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t-test, p=0.007). Data is represented as mean ± SEM and comes from n=3 mice for each condition. Scale bars are 150 µm.

    Techniques Used: Expressing, Injection, Slice Preparation, Fluorescence

    a ) Representative images showing labeling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or BG-JF 549 , respectively. b ) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labeling. c ) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. d ) Summary of the proportion of 2-step bleaching steps for each labeling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p=0.0005). e-h ) Same as a-d but with SNAP-ß2AR. * indicates statistical significance (unpaired t test, p=0.008). Scale bars are 10 µm.
    Figure Legend Snippet: a ) Representative images showing labeling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or BG-JF 549 , respectively. b ) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labeling. c ) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. d ) Summary of the proportion of 2-step bleaching steps for each labeling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p=0.0005). e-h ) Same as a-d but with SNAP-ß2AR. * indicates statistical significance (unpaired t test, p=0.008). Scale bars are 10 µm.

    Techniques Used: Labeling

    snap surface block  (New England Biolabs)


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    Structured Review

    New England Biolabs snap surface block
    Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing <t>DDR1-SNAP</t> were incubated with <t>SNAP-Surface</t> <t>Alexa</t> Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "DDR1 autophosphorylation is a result of aggregation into dense clusters"

    Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-53176-4

    Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.
    Figure Legend Snippet: Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.

    Techniques Used: Expressing, Incubation, Immunostaining, Staining, Microscopy, Binding Assay

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    • snap surface  (New England Biolabs)
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    New England Biolabs snap surface
    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.
    Snap Surface, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap substrates  (New England Biolabs)
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    New England Biolabs snap substrates
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface block  (New England Biolabs)
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    New England Biolabs snap surface block
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface probes  (New England Biolabs)
    93
    New England Biolabs snap surface probes
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Surface Probes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface block 480  (New England Biolabs)
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    New England Biolabs snap surface block 480
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Surface Block 480, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.

    Journal: International Journal of Molecular Sciences

    Article Title: Choosing the Probe for Single-Molecule Fluorescence Microscopy

    doi: 10.3390/ijms232314949

    Figure Lengend Snippet: Published comparisons of dyes aspecific interactions. AL: Alexa, AT: Atto, Abb.: Abberior. SE: succinimidyl esters; HY: hydrazide; M: maleimide.

    Article Snippet: SNAP-tag technology can be used for both extra- and intracellular labeling: in the first case, using non-cell-permeable substrates such as SNAP-Surface ® (New England Biolabs), for studying, e.g., membrane receptors [ , ]; in the second case, using cell-permeable substrates such as SNAP-Cell ® (New England Biolabs) or Janelia Fluor dyes, a quite recent family of cell-permeable fluorophores for single-molecule imaging [ ] (protocols for the synthesis of SNAP-tag ligands of Janelia Fluor and subsequent labeling can be found in [ ]).

    Techniques: Imaging, Labeling, Diffusion-based Assay, Single Particle, Fluorescence, Spectroscopy, Incubation, Transfection

    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Journal: Molecular Metabolism

    Article Title: Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

    doi: 10.1016/j.molmet.2022.101541

    Figure Lengend Snippet: Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Article Snippet: SNAP substrates were purchased from NEB (SNAP-Surface® Block, S9143S; SNAP-Surface® Alexa Fluor® 546, S9132S; SNAP-Surface® Alexa Fluor® 647, S9136S).

    Techniques: Construct, Clone Assay, Western Blot, Incubation, Flow Cytometry, Labeling