clip surface 488  (New England Biolabs)


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    New England Biolabs clip surface 488
    Clip Surface 488, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 2 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 2 article reviews
    Price from $9.99 to $1999.99
    clip surface 488 - by Bioz Stars, 2022-12
    91/100 stars

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    New England Biolabs clip surface 488
    <t>CLIP-Cas9</t> 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 <t>TMR</t> 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.
    Clip Surface 488, 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 surface 488/product/New England Biolabs
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    clip surface 488 - by Bioz Stars, 2022-12
    93/100 stars
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    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

    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