clip surface488  (New England Biolabs)


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    New England Biolabs clip surface488
    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with <t>CLIP-Surface488</t> and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.
    Clip Surface488, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clip surface488/product/New England Biolabs
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    clip surface488 - by Bioz Stars, 2022-09
    93/100 stars

    Images

    1) Product Images from "Using high-throughput barcode sequencing to efficiently map connectomes"

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx292

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with CLIP-Surface488 and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.
    Figure Legend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with CLIP-Surface488 and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.

    Techniques Used: Expressing, Staining, Cross-linking Immunoprecipitation

    Optimization of SYNseq components in HEK cells. ( A ) The presynaptic components of the SYNseq system, consisting of CLIP-Nrx1B-1xnλ and a GFP encoding barcode RNA. ( B ) The postsynaptic components of SYNseq, consisting of SNAP-Nlg1AB-1xnλ and a mCherry encoding barcode RNA. ( C and D ) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining ( C ) synPRE-P expressing HEK cells with CLIP-Surface488 and ( D ) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 μm. ( E ) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or synPOST-P. ( F ) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG–PEG–biotin–PEG–BC. We show qRT-PCR analysis of three independent RNA-IP experiments and western blot analysis of a representative sample.
    Figure Legend Snippet: Optimization of SYNseq components in HEK cells. ( A ) The presynaptic components of the SYNseq system, consisting of CLIP-Nrx1B-1xnλ and a GFP encoding barcode RNA. ( B ) The postsynaptic components of SYNseq, consisting of SNAP-Nlg1AB-1xnλ and a mCherry encoding barcode RNA. ( C and D ) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining ( C ) synPRE-P expressing HEK cells with CLIP-Surface488 and ( D ) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 μm. ( E ) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or synPOST-P. ( F ) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG–PEG–biotin–PEG–BC. We show qRT-PCR analysis of three independent RNA-IP experiments and western blot analysis of a representative sample.

    Techniques Used: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    2) Product Images from "A ligand-based system for receptor-specific delivery of proteins"

    Article Title: A ligand-based system for receptor-specific delivery of proteins

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55797-1

    CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.
    Figure Legend Snippet: CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.

    Techniques Used: Cross-linking Immunoprecipitation, Activity Assay, Electroporation, Staining

    Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).
    Figure Legend Snippet: Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation, Expressing, Staining

    3) Product Images from "Using high-throughput barcode sequencing to efficiently map connectomes"

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    Journal: bioRxiv

    doi: 10.1101/099093

    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.
    Figure Legend Snippet: Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Techniques Used: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.
    Figure Legend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Techniques Used: Expressing, Staining, Cross-linking Immunoprecipitation

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    New England Biolabs clip surface 488
    CTR1 co-localization with membrane or endosome markers after Cu treatment. (A) <t>HEK-CLIP–CTR1</t> cells were incubated with <t>CLIP-Surface–488</t> (green), followed by treatment with or without 30 µM Cu and with fluorescent transferrin
    Clip Surface 488, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clip surface 488/product/New England Biolabs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    clip surface 488 - by Bioz Stars, 2022-09
    91/100 stars
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    New England Biolabs clip surface488
    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with <t>CLIP-Surface488</t> and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.
    Clip Surface488, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clip surface488/product/New England Biolabs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    clip surface488 - by Bioz Stars, 2022-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    CTR1 co-localization with membrane or endosome markers after Cu treatment. (A) HEK-CLIP–CTR1 cells were incubated with CLIP-Surface–488 (green), followed by treatment with or without 30 µM Cu and with fluorescent transferrin

    Journal: Journal of Cell Science

    Article Title: Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu+ uptake system

    doi: 10.1242/jcs.173351

    Figure Lengend Snippet: CTR1 co-localization with membrane or endosome markers after Cu treatment. (A) HEK-CLIP–CTR1 cells were incubated with CLIP-Surface–488 (green), followed by treatment with or without 30 µM Cu and with fluorescent transferrin

    Article Snippet: Antibodies used for immunofluorescence were diluted at 1:1000; rabbit anti-EEA1 (ab2900; Abcam, Cambridge, MA, USA), mouse anti-transferrin-receptor (TfR; ab84036; Abcam), mouse anti-HA (sc-7392; Santa Cruz, Dallas, TX, USA), mouse anti-FLAG ( ; Genscript), mouse anti-Rab9 (MA3-067; Affinity Bioreagents, Golden, CO, USA), mouse anti-Rab7 (ab50533; Abcam), mouse anti-Rab11 (ab170134; Abcam), rabbit anti-Rab4 (PA3-912; Thermo Scientific, Rockford, IL, USA), and Surface-CLIP–547 or –488 (S9233S, S9232S; New England Biolabs, Ipswich, MA, USA).

    Techniques: Cross-linking Immunoprecipitation, Incubation

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with CLIP-Surface488 and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.

    Journal: Nucleic Acids Research

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1093/nar/gkx292

    Figure Lengend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the ( A ) presynaptic or ( B ) postsynaptic components of SYNseq. ( C and D ) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining ( C ) synPRE-P with CLIP-Surface488 and ( D ) synPOST-P with SNAP-Surface488. Scale bar = 5 μm.

    Article Snippet: Tagging with BG/BC derivatives We obtained all BG- and BC-functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer's instructions.

    Techniques: Expressing, Staining, Cross-linking Immunoprecipitation

    Optimization of SYNseq components in HEK cells. ( A ) The presynaptic components of the SYNseq system, consisting of CLIP-Nrx1B-1xnλ and a GFP encoding barcode RNA. ( B ) The postsynaptic components of SYNseq, consisting of SNAP-Nlg1AB-1xnλ and a mCherry encoding barcode RNA. ( C and D ) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining ( C ) synPRE-P expressing HEK cells with CLIP-Surface488 and ( D ) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 μm. ( E ) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or synPOST-P. ( F ) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG–PEG–biotin–PEG–BC. We show qRT-PCR analysis of three independent RNA-IP experiments and western blot analysis of a representative sample.

    Journal: Nucleic Acids Research

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1093/nar/gkx292

    Figure Lengend Snippet: Optimization of SYNseq components in HEK cells. ( A ) The presynaptic components of the SYNseq system, consisting of CLIP-Nrx1B-1xnλ and a GFP encoding barcode RNA. ( B ) The postsynaptic components of SYNseq, consisting of SNAP-Nlg1AB-1xnλ and a mCherry encoding barcode RNA. ( C and D ) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining ( C ) synPRE-P expressing HEK cells with CLIP-Surface488 and ( D ) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 μm. ( E ) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or synPOST-P. ( F ) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG–PEG–biotin–PEG–BC. We show qRT-PCR analysis of three independent RNA-IP experiments and western blot analysis of a representative sample.

    Article Snippet: Tagging with BG/BC derivatives We obtained all BG- and BC-functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer's instructions.

    Techniques: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.

    Journal: Scientific Reports

    Article Title: A ligand-based system for receptor-specific delivery of proteins

    doi: 10.1038/s41598-019-55797-1

    Figure Lengend Snippet: CLIP-Cas9 activity and internalization in keratinocytes. ( a ) Schematic of CLIP-Cas9::sgRNA electroporation strategy. ( b ) Indel spectrum determined by TIDE of primary keratinocytes electroporated with CLIP-Cas9::sgRNA targeting the Atat1 gene. The inset show T7 endonuclease 1 assay performed on genomic DNA from electroporated keratinocytes. t.e. = total efficiency. ( c ) Quantification (% cells) and representative images ( d ) of TMR positive cells upon 2 hours treatment with 2 μM of ligand cross-linked Cas9 (#1 no sgRNA; #2 with sgRNA; #3 with sgRNA+ protamine; #4 with sgRNA + ppTG21). Nuclei were stained with Hoechst. Scale bars, 20 μm. The horizontal lines mark the geometric mean and the error bars mark the standard error.

    Article Snippet: SDS-PAGE and western blotting To assess the coupling reaction, CLIP-Cre or CLIP-Cas9 were coupled with an excess of BC-Surface488 or BCTMR at a 1:1.5 molar ratio (NEB #S9232S; #S9219S) for 1 hour at 37 °C in PBS (pH 7.4).

    Techniques: Cross-linking Immunoprecipitation, Activity Assay, Electroporation, Staining

    Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).

    Journal: Scientific Reports

    Article Title: A ligand-based system for receptor-specific delivery of proteins

    doi: 10.1038/s41598-019-55797-1

    Figure Lengend Snippet: Binding of SNAP-tagged ligands to keratinocytes and selective cross-linking to CLIP-tagged enzymes. ( a ) Schematic representation of one keratinocyte expressing the receptors of interest and the ligands used. ( b ) Quantification of labelled IL-31 K138A SNAP-BG 549 ( c ), NGF R121W SNAP-BG 549 ( d ) and SNAP-BG 549 (green bars) binding to primary keratinocytes. Nuclear localization was observed after 2 hours treatment. The nuclei were stained with Hoechst. Scale bars, 20 μm. The insets represent corresponding brightfield images. ( e ) 3D structures showing selective cross-linking of SNAP-tagged ligands (NGF-SNAP) and CLIP-tagged proteins (CLIP-Cre) through a BG-TMR-PEG-BC linker (PDB ID codes: 1BET, 1KBU, 3KZY). ( f ) Schematic representation of S-CROSS optimized chemical reaction. ( g ) Representative Coomassie gel showing cross-linking complexes (red asterisks). First lane (#1) is IL-31 SNAP::CLIP CRE, second lane (#2) is NGF SNAP::CLIP CRE and third lane (#3) is SNAP::CLIP-CRE. ( h ) Quantification of cross-linking from Coomassie gel ( g ).

    Article Snippet: SDS-PAGE and western blotting To assess the coupling reaction, CLIP-Cre or CLIP-Cas9 were coupled with an excess of BC-Surface488 or BCTMR at a 1:1.5 molar ratio (NEB #S9232S; #S9219S) for 1 hour at 37 °C in PBS (pH 7.4).

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Expressing, Staining

    Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: Optimization of SYNseq components in HEK cells. (a) The presynaptic components of the SYNseq system, consisting of CLIP-NRXN-1xnλ and a GFP encoding barcode RNA. (b) The postsynaptic components of SYNseq, consisting of SNAP-NLGN-1xnλ and a mCherry encoding barcode RNA. (c,d) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining (c) synPRE-P expressing HEK cells with CLIP-Surface488 and (d) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 µm. (e) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or POST. (f) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG-PEG-Biotin-PEG-BC. We show qRT-PCR analysis of 3 independent RNA-IP experiments and western blot analysis of a representative sample.

    Article Snippet: Tagging with BG/BC derivativesWe obtained all BG- and BC- functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer’s instructions.

    Techniques: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR

    Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Journal: bioRxiv

    Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

    doi: 10.1101/099093

    Figure Lengend Snippet: Viral expression and membrane trafficking of synPRE-P and synPOST-P in neurons. We use a double promoter Sindbis virus that expresses the (a) presynaptic or (b) postsynaptic components of SYNseq. (c,d) When expressed in neurons using Sindbis virus synPRE-P and synPOST-P show clear membrane trafficking as revealed by staining (c) synPRE-P with CLIP-Surface488 and (d) synPOST-P with SNAP-Surface488. Scale bar = 5 µ m.

    Article Snippet: Tagging with BG/BC derivativesWe obtained all BG- and BC- functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer’s instructions.

    Techniques: Expressing, Staining, Cross-linking Immunoprecipitation