streptavidin-coated beads Search Results


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  • 99
    New England Biolabs streptavidin coated beads
    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S <t>streptavidin,</t> M magnetic bead.
    Streptavidin Coated Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher streptavidin coated paramagnetic beads
    Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a <t>streptavidin</t> matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.
    Streptavidin Coated Paramagnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega streptavidin coated beads
    RNA capture assay. (A) Flow chart of the RNA capture assay. Genetically distinct RNA molecules are represented as A (thick line) and B (thin line). RNA dimers were annealed to a biotinylated oligonucleotide before incubation on <t>streptavidin-coated</t> beads.
    Streptavidin Coated Beads, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher streptavidin coated beads
    Magnetic tweezers set-up for measuring on a tethered DNA molecule. a Schematic of the set-up. A LED illuminates the flow cell through a lens and the magnet holder. Imaging is done with a 50x Nikon objective onto a CCD camera. Magnets manipulate a magnetic bead attached to the DNA. b A flow cell is constructed with 24x60mm coverslips. The bottom coverslip is amine-coated and has reference beads bound to it. The top coverslip has sandblasted holes to allow fluid flow. Parafilm is used to seal the coverslips and to create a ˜50 μl flow cell volume. c Schematic of a tethered DNA molecule. A DNA molecule is linked to a <t>streptavidin-coated</t> magnetic bead with biotin, and to azide groups on the surface with DBCO at the other end
    Streptavidin Coated Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 699 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare streptavidin coated beads
    GrA binds several Gram-negative bacteria but not LPS. ( a ) GrA binds to Gram-negative bacteria. GrA-biotin binding to E. coli BL21 , E. coli ATCC 25922 , E. coli Expec 536 , P. aeruginosa (PA-01), or NM HB-1 (NM) was detected by flow cytometry. Data are representative of at least three independent experiments. ( b ) GrA does not bind to LPS in a solid-phase binding assay. LPS was immobilized and incubated with biotinylated GrK, GrA, or GrA-SA. Data are depicted as specific binding (depicted as % of maximum GrK binding) and represent mean±S.D. of three independent experiments. ( c ) GrA does not bind to LPS in a pull-down assay. LPS-biotin was coupled to <t>streptavidin-sepharose</t> beads. After washing, beads were incubated with GrA (upper panel) or GrA-SA (lower panel). GrK was used as a positive control. Bound protein was eluted from the beads and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by total protein staining. Results are representative for three independent experiments. ( d ) GrA does not bind to LPS in a surface plasmon resonance assay. Immobilized GrA and GrK were incubated with LPS for 10 min. (association), followed by a buffer flow (dissociation). The association of LPS to GrA and GrK at t =10 min is shown. Results represent mean±S.D. of three independent experiments. RU, response units. ( e ) Direct comparison of LPS (100 μ g/ml) binding with immobilized GrK and GrA. Results are representative of three independent experiments. RU, response units.
    Streptavidin Coated Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche streptavidin coated beads
    GrA binds several Gram-negative bacteria but not LPS. ( a ) GrA binds to Gram-negative bacteria. GrA-biotin binding to E. coli BL21 , E. coli ATCC 25922 , E. coli Expec 536 , P. aeruginosa (PA-01), or NM HB-1 (NM) was detected by flow cytometry. Data are representative of at least three independent experiments. ( b ) GrA does not bind to LPS in a solid-phase binding assay. LPS was immobilized and incubated with biotinylated GrK, GrA, or GrA-SA. Data are depicted as specific binding (depicted as % of maximum GrK binding) and represent mean±S.D. of three independent experiments. ( c ) GrA does not bind to LPS in a pull-down assay. LPS-biotin was coupled to <t>streptavidin-sepharose</t> beads. After washing, beads were incubated with GrA (upper panel) or GrA-SA (lower panel). GrK was used as a positive control. Bound protein was eluted from the beads and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by total protein staining. Results are representative for three independent experiments. ( d ) GrA does not bind to LPS in a surface plasmon resonance assay. Immobilized GrA and GrK were incubated with LPS for 10 min. (association), followed by a buffer flow (dissociation). The association of LPS to GrA and GrK at t =10 min is shown. Results represent mean±S.D. of three independent experiments. RU, response units. ( e ) Direct comparison of LPS (100 μ g/ml) binding with immobilized GrK and GrA. Results are representative of three independent experiments. RU, response units.
    Streptavidin Coated Beads, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Spherotech streptavidin coated beads
    DNA dumbbell construction. Step-by-step process of DNA dumbbell construction and exposure to intercalating dye in a flow-free environment using reservoir-based LFC. (1) Individual trapping of DNA-attached <t>streptavidin</t> coated (diameter: 1.76 µm) and free anti-digoxigenin coated beads (diameter: 0.9 µm) within the reservoir and translocating into the main channel; (top-right inset: bright-field image of the trapped beads) (2) applying gentle flow (0.05 mm/s) for a few seconds to elongate DNA and attachment to second bead (3) Incubation of the DNA dumbbell with YOYO-1 in a flow-free reservoir. (4) Visualization of the DNA in the flow-free main channel; bottom-left inset: fluorescent image of DNA dumbbell.
    Streptavidin Coated Beads, supplied by Spherotech, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bangs Laboratories streptavidin coated beads
    Quality control of explant biotinylation. A. Detection of biotin using Steptavidin-FITC on control explant cultures (A a , A b , no biotin) or biotinylated cultures (A c , A d ). Explants were also treated with an α-NCAM antibody detected with red fluorescence to label axons (A b , A d ) and Hoescht to label cell nuclei (blue, A b , A d ). A a and A c show the <t>streptavidin</t> labeling only, while A b and A d show the triple labeling. B. High magnification of a biotinylated axon, showing patches of biotinylation along the axon (green fluorescence), and a glass microbead (square) attached to a large patch. The bead is better seen in the inset.
    Streptavidin Coated Beads, supplied by Bangs Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    PolyScience streptavidin coated beads
    Quality control of explant biotinylation. A. Detection of biotin using Steptavidin-FITC on control explant cultures (A a , A b , no biotin) or biotinylated cultures (A c , A d ). Explants were also treated with an α-NCAM antibody detected with red fluorescence to label axons (A b , A d ) and Hoescht to label cell nuclei (blue, A b , A d ). A a and A c show the <t>streptavidin</t> labeling only, while A b and A d show the triple labeling. B. High magnification of a biotinylated axon, showing patches of biotinylation along the axon (green fluorescence), and a glass microbead (square) attached to a large patch. The bead is better seen in the inset.
    Streptavidin Coated Beads, supplied by PolyScience, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ChromoTek streptavidin coated beads
    In vivo biotinylation of GTA-tagged protein and removal of endogenously biotinylated proteins by Tobacco etch virus (TEV) cleavage. a Western blot detection of biotinylated proteins in lysed wild-type N2 animals ( left lane ) and lysed animals expressing GTA-tagged DLG-1 and ubiquitously expressed BirA ( right lane ). An additional band of the correct estimated molecular weight for DLG-1::GTA is detected upon expression of BirA ( arrow ). b Western blot analysis of eluate and beads after purification and release of bound bait protein by TEV cleavage. Both blots contain the same samples. The background of naturally biotinylated proteins remains bound to the <t>streptavidin</t> beads and is visible on the α-biotin western blot in the beads remainder, while tagged DLG-1 is cleaved off and visible on the α-GFP western blot in the eluate. No DLG-1 protein is purified in samples from animals not expressing BirA. c Lanes 1 and 2 : Western blot analysis of the levels of GTA::CDC-42 purified with GFP-Trap beads ( lane 1 ) and streptavidin beads ( lane 2 ) from animals expressing BirA in seam and hyp7 epidermal cells. Lanes 3 and 4 : Western blot analysis of beads ( lane 3 ) and eluate ( lane 4 ) after cleavage of GTA::CDC-42 purified with streptavidin beads. d Schematic of the TEV cleavage procedure
    Streptavidin Coated Beads, supplied by ChromoTek, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Spherotech streptavidin coated bead
    DNA hairpin substrate design. Schematic representation of the DNA hairpin substrate extended between two trapped beads (not to scale). The construct consisted of three separate fragments ligated together: a 1550-bp ‘Left handle’ (L; cyan), a 1500-bp ‘Right handle’ (R; dark blue), and the 89-bp (with (dT) 4 tetraloop) hairpin (red). L and R served as linkers attached to the trapped beads via 5′-biotin and 5′-digoxigenin modifications, respectively. L was attached to a 0.79-μm <t>streptavidin-coated</t> bead, R to a 0.86-μm anti-digoxigenin-coated bead. The hairpin was flanked at the 5′ end by a (dT) n ssDNA region that serves as a helicase loading site (here n = 10) and at the 3′ end by an abasic site (dark blue open circle). The construct was modular, allowing the hairpin sequence and the loading site length to be varied (see ‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.004
    Streptavidin Coated Bead, supplied by Spherotech, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Spherotec streptavidin coated bead
    DNA hairpin substrate design. Schematic representation of the DNA hairpin substrate extended between two trapped beads (not to scale). The construct consisted of three separate fragments ligated together: a 1550-bp ‘Left handle’ (L; cyan), a 1500-bp ‘Right handle’ (R; dark blue), and the 89-bp (with (dT) 4 tetraloop) hairpin (red). L and R served as linkers attached to the trapped beads via 5′-biotin and 5′-digoxigenin modifications, respectively. L was attached to a 0.79-μm <t>streptavidin-coated</t> bead, R to a 0.86-μm anti-digoxigenin-coated bead. The hairpin was flanked at the 5′ end by a (dT) n ssDNA region that serves as a helicase loading site (here n = 10) and at the 3′ end by an abasic site (dark blue open circle). The construct was modular, allowing the hairpin sequence and the loading site length to be varied (see ‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.004
    Streptavidin Coated Bead, supplied by Spherotec, used in various techniques. Bioz Stars score: 89/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore streptavidin coated beads
    CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. (A) Cell lysates of LPS-primed BMDMs were incubated with different concentrations of biotin–CY-09, which were then pulled down with <t>streptavidin</t> beads. (B) Cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells were incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (C) Purified human NLRP3 protein was incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) Purified human NLRP3 protein was incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (E) MST assay for the affinity between CY-09 and purified GFP-NLRP3 protein. (F and G) Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, AIM2, NLRC4, NLRP3–LRR, NLPR3–NACHT, or NLRP3–PYD constructs were incubated with indicated concentration of biotin–CY-09, which were then pulled down with streptavidin beads. Data are representative of three independent experiments.
    Streptavidin Coated Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 146 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    PerkinElmer streptavidin coated spa beads
    AdoHcy binds the DENV3 MTase with a much weaker affinity than do AdoMet and SIN. ( A ) Dose response of inhibition of the [ 3 H]-SAM-MTase complex formation by AdoMet (black), AdoHcy (red), and SIN (green). The biotinylated DENV3 MTase and 3 H-labeled SAM were incubated with or without compounds AdoMet, AdoHcy, and SIN. A two-fold dilution series was shown for each compound. The reaction mixtures were mixed with the <t>streptavidin-coated</t> <t>SPA</t> beads and quantified using a Microbeta 2 scintillation counter. ( B ). Superposition of the crystal structures of the MTase-SIN complex (green) [ 23 ] and the MTase-SAH complex (yellow) [ 19 ]. SAH and SIN are shown in stick representation. Atomic color coding is as follows (unless otherwise specified): carbon in yellow/green, oxygen in red, nitrogen in blue, and sulfur in orange. Potential hydrogen bonds are depicted in red dashed lines.
    Streptavidin Coated Spa Beads, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated spa beads/product/PerkinElmer
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    91
    Merck & Co streptavidin coated paramagnetic beads
    AdoHcy binds the DENV3 MTase with a much weaker affinity than do AdoMet and SIN. ( A ) Dose response of inhibition of the [ 3 H]-SAM-MTase complex formation by AdoMet (black), AdoHcy (red), and SIN (green). The biotinylated DENV3 MTase and 3 H-labeled SAM were incubated with or without compounds AdoMet, AdoHcy, and SIN. A two-fold dilution series was shown for each compound. The reaction mixtures were mixed with the <t>streptavidin-coated</t> <t>SPA</t> beads and quantified using a Microbeta 2 scintillation counter. ( B ). Superposition of the crystal structures of the MTase-SIN complex (green) [ 23 ] and the MTase-SAH complex (yellow) [ 19 ]. SAH and SIN are shown in stick representation. Atomic color coding is as follows (unless otherwise specified): carbon in yellow/green, oxygen in red, nitrogen in blue, and sulfur in orange. Potential hydrogen bonds are depicted in red dashed lines.
    Streptavidin Coated Paramagnetic Beads, supplied by Merck & Co, 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/streptavidin coated paramagnetic beads/product/Merck & Co
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    85
    Thermo Fisher streptavidin coated dyna beads
    AdoHcy binds the DENV3 MTase with a much weaker affinity than do AdoMet and SIN. ( A ) Dose response of inhibition of the [ 3 H]-SAM-MTase complex formation by AdoMet (black), AdoHcy (red), and SIN (green). The biotinylated DENV3 MTase and 3 H-labeled SAM were incubated with or without compounds AdoMet, AdoHcy, and SIN. A two-fold dilution series was shown for each compound. The reaction mixtures were mixed with the <t>streptavidin-coated</t> <t>SPA</t> beads and quantified using a Microbeta 2 scintillation counter. ( B ). Superposition of the crystal structures of the MTase-SIN complex (green) [ 23 ] and the MTase-SAH complex (yellow) [ 19 ]. SAH and SIN are shown in stick representation. Atomic color coding is as follows (unless otherwise specified): carbon in yellow/green, oxygen in red, nitrogen in blue, and sulfur in orange. Potential hydrogen bonds are depicted in red dashed lines.
    Streptavidin Coated Dyna Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Journal: Biological Procedures Online

    Article Title: A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

    doi: 10.1007/s12575-009-9022-z

    Figure Lengend Snippet: General scheme applied for identifying peanut immature pod-specific genes (tracer mRNA (1)) after a single round subtraction . B biotin, S streptavidin, M magnetic bead.

    Article Snippet: Streptavidin-coated magnetic beads (1.1 mg/ml) were applied again to purify synthesized double-stranded cDNAs with three times washes.

    Techniques:

    Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Selection of streptavidin-binding DeNAno. ( A ) Schematic of selection process. ( B ) Staining of streptavidin selection rounds 1–5. Probe-only, library and positive control (biotinylated library) are also shown. ( C ) Staining of four selected streptavidin clones on streptavidin beads and BSA-coated beads. Negative control clone (G10neg) and biotinylated positive control clone (G10bio) also shown.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Selection, Binding Assay, Staining, Positive Control, Clone Assay, Negative Control

    Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Competitive titration and competitive release of streptavidin-binding DeNAno with biotin/biotin derivatives or streptavidin. ( A ) Schematic of competitive titration using biotin/biotin derivatives. ( B ) Free biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competition titrations were done by pre-incubating streptavidin beads with one of the biotin/biotin derivatives (or buffer for the baseline), then adding DeNAno particles. ( C ) Schematic of competitive release using biotin/biotin derivatives. ( D ) Biotin (top), desthiobiotin (middle) and 2-iminobiotin (bottom) competitive release assays were done by staining streptavidin beads with DeNAno particles, then adding biotin/biotin derivative (or buffer for baseline). ( E ) Schematic of streptavidin competitive titration. ( F ) Free streptavidin competition titration of SA-D7 and SA-D8 clones and G10bio positive control. Fluorescently-labeled DeNAno particles were pre-incubated with varying concentrations of free streptavidin, then streptavidin beads were added.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Titration, Binding Assay, Staining, Clone Assay, Positive Control, Labeling, Incubation

    Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Dissociation of streptavidin-binding DeNAno over time. Streptavidin-coated magnetic beads were stained with DeNAno particles. The stained beads were then incubated in 10 ml buffer for 35 days. Aliquots were taken every week of the total sample (supernatant plus beads) and supernatant only (beads were removed by magnet). PCR was done on all samples/timepoints and percentage release is graphed (DeNAno in supernatant/DeNAno in total * 100%).

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Binding Assay, Magnetic Beads, Staining, Incubation, Polymerase Chain Reaction

    Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Journal: Nucleic Acids Research

    Article Title: Selection of DNA nanoparticles with preferential binding to aggregated protein target

    doi: 10.1093/nar/gkw136

    Figure Lengend Snippet: Imaging of DeNAno and staining different size DeNAno. ( A ) Atomic force micrograph (AFM) of dried DeNAno SA-D8 on poly-L-lysine-coated mica. Scale = 400 nm. ( B ) SA-D8 DeNAno roughly 75 nm in diameter as observed by transmission electron microscopy (TEM) using negative staining. Scale = 100 nm. ( C ) TEM of small SA-D8 DeNAno roughly 58 nm in diameter. Scale = 100 nm. ( D ) Binding of streptavidin DeNAno of different sizes made by alteration of dNTP concentration. DeNAno particles were made with 3 nmol dNTPs for 30 m at 30°C (the standard conditions), or 93.8 pmol dNTPs for 30 m at 30°C. A control DeNAno from a different library was also made for both of these conditions and used as an internal control in the staining and subsequent PCR. The ratio of the bound particles (streptavidin DeNAno:control DeNano) to total particles (streptavidin DeNAno:control DeNAno) is graphed.

    Article Snippet: Beads Streptavidin-coated magnetic beads (NEB) were used for selections/staining for streptavidin-specific DeNAno with no modifications.

    Techniques: Imaging, Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Negative Staining, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.

    Journal: BMC Genomics

    Article Title: Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells

    doi: 10.1186/s12864-015-1489-1

    Figure Lengend Snippet: Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.

    Article Snippet: Reverse-transcribed cDNAs were random-fragmented (Bioruptor, Diagenode) to an average size of approximately 200 nucleotides, and subsequently enriched for 3′ fragments through binding to streptavidin-coated paramagnetic beads (Dynabeads M-270 Streptavidin, Invitrogen).

    Techniques: Generated, Sequencing, Functional Assay, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Purification, Derivative Assay, Expressing

    RNA capture assay. (A) Flow chart of the RNA capture assay. Genetically distinct RNA molecules are represented as A (thick line) and B (thin line). RNA dimers were annealed to a biotinylated oligonucleotide before incubation on streptavidin-coated beads.

    Journal:

    Article Title: Nonrandom Dimerization of Murine Leukemia Virus Genomic RNAs

    doi: 10.1128/JVI.78.22.12129-12139.2004

    Figure Lengend Snippet: RNA capture assay. (A) Flow chart of the RNA capture assay. Genetically distinct RNA molecules are represented as A (thick line) and B (thin line). RNA dimers were annealed to a biotinylated oligonucleotide before incubation on streptavidin-coated beads.

    Article Snippet: Cooled oligonucleotide-bound RNA was immediately added to streptavidin-coated beads (Promega; prewashed with 0.5× SSC three times and suspended in 100 μl of 0.5× SSC) and gently mixed for 10 min at room temperature.

    Techniques: Flow Cytometry, Incubation

    Pull-down of biotinylated targets from spiked HeLa cell lysate by affinity-matured monobodies. Four biotinylated targets were mixed (1 pmol each) with HeLa cell lysate (10 µg). FLAG-tagged FN3 monobodies were added to the mixture, and subsequent target/monobody complex was pulled down via anti-FLAG antibody-coated paramagnetic beads. After washing, the target/monobody complex was eluted off the beads and used in Western blot. Biotinylated targets were detected via a streptavidin-infrared dye conjugate. Red arrows indicate the positions of two pull-downed targets on the blot.

    Journal: International Journal of Molecular Sciences

    Article Title: Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    doi: 10.3390/ijms161023587

    Figure Lengend Snippet: Pull-down of biotinylated targets from spiked HeLa cell lysate by affinity-matured monobodies. Four biotinylated targets were mixed (1 pmol each) with HeLa cell lysate (10 µg). FLAG-tagged FN3 monobodies were added to the mixture, and subsequent target/monobody complex was pulled down via anti-FLAG antibody-coated paramagnetic beads. After washing, the target/monobody complex was eluted off the beads and used in Western blot. Biotinylated targets were detected via a streptavidin-infrared dye conjugate. Red arrows indicate the positions of two pull-downed targets on the blot.

    Article Snippet: After 2 h tumbling at room temperature, the streptavidin-coated paramagnetic beads were captured with a magnet and washed three times with PBS plus 0.1% Tween 20, and then another two washes with PBS.

    Techniques: Western Blot

    hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic

    Journal: The Journal of Biological Chemistry

    Article Title: Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1? with the Nucleosome *

    doi: 10.1074/jbc.M112.390849

    Figure Lengend Snippet: hHP1β specifically interacts with H3K9me3 nucleosomes. A and B, pulldown experiments using synthetic nucleosomes uniformly containing the indicated histone H3 modification status immobilized via biotinylated DNA on streptavidin-coated magnetic

    Article Snippet: 1 μg of biotinylated nucleosomes (reconstituted with DNA ligated via an EcoRI site to a biotinylated oligonucleotide) were preincubated with 50 μg of streptavidin-coated magnetic beads (Promega) in binding buffer (10 m m triethanolamine-HCl, 150 m m NaCl, 0.1% v/v Triton X-100, 5% v/v glycerol, 0.1 m m EDTA, pH 7.5) for 4 h at 4 °C.

    Techniques: Modification

    Magnetic tweezers set-up for measuring on a tethered DNA molecule. a Schematic of the set-up. A LED illuminates the flow cell through a lens and the magnet holder. Imaging is done with a 50x Nikon objective onto a CCD camera. Magnets manipulate a magnetic bead attached to the DNA. b A flow cell is constructed with 24x60mm coverslips. The bottom coverslip is amine-coated and has reference beads bound to it. The top coverslip has sandblasted holes to allow fluid flow. Parafilm is used to seal the coverslips and to create a ˜50 μl flow cell volume. c Schematic of a tethered DNA molecule. A DNA molecule is linked to a streptavidin-coated magnetic bead with biotin, and to azide groups on the surface with DBCO at the other end

    Journal: BMC Biophysics

    Article Title: Copper-free click chemistry for attachment of biomolecules in magnetic tweezers

    doi: 10.1186/s13628-015-0023-9

    Figure Lengend Snippet: Magnetic tweezers set-up for measuring on a tethered DNA molecule. a Schematic of the set-up. A LED illuminates the flow cell through a lens and the magnet holder. Imaging is done with a 50x Nikon objective onto a CCD camera. Magnets manipulate a magnetic bead attached to the DNA. b A flow cell is constructed with 24x60mm coverslips. The bottom coverslip is amine-coated and has reference beads bound to it. The top coverslip has sandblasted holes to allow fluid flow. Parafilm is used to seal the coverslips and to create a ˜50 μl flow cell volume. c Schematic of a tethered DNA molecule. A DNA molecule is linked to a streptavidin-coated magnetic bead with biotin, and to azide groups on the surface with DBCO at the other end

    Article Snippet: Streptavidin-coated beads (M270 Streptavidin coated, Life Technologies) were incubated with the biotin-functionalized DNA for 20 min. After incubation, the beads were washed 3 times with washing buffer with 0.05 % Tween.

    Techniques: Flow Cytometry, Imaging, Construct

    GrA binds several Gram-negative bacteria but not LPS. ( a ) GrA binds to Gram-negative bacteria. GrA-biotin binding to E. coli BL21 , E. coli ATCC 25922 , E. coli Expec 536 , P. aeruginosa (PA-01), or NM HB-1 (NM) was detected by flow cytometry. Data are representative of at least three independent experiments. ( b ) GrA does not bind to LPS in a solid-phase binding assay. LPS was immobilized and incubated with biotinylated GrK, GrA, or GrA-SA. Data are depicted as specific binding (depicted as % of maximum GrK binding) and represent mean±S.D. of three independent experiments. ( c ) GrA does not bind to LPS in a pull-down assay. LPS-biotin was coupled to streptavidin-sepharose beads. After washing, beads were incubated with GrA (upper panel) or GrA-SA (lower panel). GrK was used as a positive control. Bound protein was eluted from the beads and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by total protein staining. Results are representative for three independent experiments. ( d ) GrA does not bind to LPS in a surface plasmon resonance assay. Immobilized GrA and GrK were incubated with LPS for 10 min. (association), followed by a buffer flow (dissociation). The association of LPS to GrA and GrK at t =10 min is shown. Results represent mean±S.D. of three independent experiments. RU, response units. ( e ) Direct comparison of LPS (100 μ g/ml) binding with immobilized GrK and GrA. Results are representative of three independent experiments. RU, response units.

    Journal: Cell Death Discovery

    Article Title: Granzymes A and K differentially potentiate LPS-induced cytokine response

    doi: 10.1038/cddiscovery.2016.84

    Figure Lengend Snippet: GrA binds several Gram-negative bacteria but not LPS. ( a ) GrA binds to Gram-negative bacteria. GrA-biotin binding to E. coli BL21 , E. coli ATCC 25922 , E. coli Expec 536 , P. aeruginosa (PA-01), or NM HB-1 (NM) was detected by flow cytometry. Data are representative of at least three independent experiments. ( b ) GrA does not bind to LPS in a solid-phase binding assay. LPS was immobilized and incubated with biotinylated GrK, GrA, or GrA-SA. Data are depicted as specific binding (depicted as % of maximum GrK binding) and represent mean±S.D. of three independent experiments. ( c ) GrA does not bind to LPS in a pull-down assay. LPS-biotin was coupled to streptavidin-sepharose beads. After washing, beads were incubated with GrA (upper panel) or GrA-SA (lower panel). GrK was used as a positive control. Bound protein was eluted from the beads and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by total protein staining. Results are representative for three independent experiments. ( d ) GrA does not bind to LPS in a surface plasmon resonance assay. Immobilized GrA and GrK were incubated with LPS for 10 min. (association), followed by a buffer flow (dissociation). The association of LPS to GrA and GrK at t =10 min is shown. Results represent mean±S.D. of three independent experiments. RU, response units. ( e ) Direct comparison of LPS (100 μ g/ml) binding with immobilized GrK and GrA. Results are representative of three independent experiments. RU, response units.

    Article Snippet: LPS-Granzyme pull-down assay LPS-biotin (50 μ g/ml) was coupled to streptavidin-coated beads (Amersham Biosciences, GE Healthcare, Chicago, IL, USA).

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Incubation, Pull Down Assay, Positive Control, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, SPR Assay

    DNA dumbbell construction. Step-by-step process of DNA dumbbell construction and exposure to intercalating dye in a flow-free environment using reservoir-based LFC. (1) Individual trapping of DNA-attached streptavidin coated (diameter: 1.76 µm) and free anti-digoxigenin coated beads (diameter: 0.9 µm) within the reservoir and translocating into the main channel; (top-right inset: bright-field image of the trapped beads) (2) applying gentle flow (0.05 mm/s) for a few seconds to elongate DNA and attachment to second bead (3) Incubation of the DNA dumbbell with YOYO-1 in a flow-free reservoir. (4) Visualization of the DNA in the flow-free main channel; bottom-left inset: fluorescent image of DNA dumbbell.

    Journal: Scientific Reports

    Article Title: Additive manufacturing of laminar flow cells for single-molecule experiments

    doi: 10.1038/s41598-019-53151-z

    Figure Lengend Snippet: DNA dumbbell construction. Step-by-step process of DNA dumbbell construction and exposure to intercalating dye in a flow-free environment using reservoir-based LFC. (1) Individual trapping of DNA-attached streptavidin coated (diameter: 1.76 µm) and free anti-digoxigenin coated beads (diameter: 0.9 µm) within the reservoir and translocating into the main channel; (top-right inset: bright-field image of the trapped beads) (2) applying gentle flow (0.05 mm/s) for a few seconds to elongate DNA and attachment to second bead (3) Incubation of the DNA dumbbell with YOYO-1 in a flow-free reservoir. (4) Visualization of the DNA in the flow-free main channel; bottom-left inset: fluorescent image of DNA dumbbell.

    Article Snippet: Streptavidin-coated beads (diameter ~1.76 µm, Spherotech) with concentration of 0.01 mg/ml were incubated with a digoxigenin-biotin-tagged 12 kb fragment of λ-DNA with a concentration of 0.4 ng/μl in 500 μl PBS buffer for 1 hour.

    Techniques: Flow Cytometry, Incubation

    Quality control of explant biotinylation. A. Detection of biotin using Steptavidin-FITC on control explant cultures (A a , A b , no biotin) or biotinylated cultures (A c , A d ). Explants were also treated with an α-NCAM antibody detected with red fluorescence to label axons (A b , A d ) and Hoescht to label cell nuclei (blue, A b , A d ). A a and A c show the streptavidin labeling only, while A b and A d show the triple labeling. B. High magnification of a biotinylated axon, showing patches of biotinylation along the axon (green fluorescence), and a glass microbead (square) attached to a large patch. The bead is better seen in the inset.

    Journal: Bio-protocol

    Article Title: Preparation and Manipulation of Olfactory Epithelium Explant Cultures for Measurement of the Mechanical Tension of Individual Axons Using the Biomembrane Force Probe

    doi: 10.21769/BioProtoc.3213

    Figure Lengend Snippet: Quality control of explant biotinylation. A. Detection of biotin using Steptavidin-FITC on control explant cultures (A a , A b , no biotin) or biotinylated cultures (A c , A d ). Explants were also treated with an α-NCAM antibody detected with red fluorescence to label axons (A b , A d ) and Hoescht to label cell nuclei (blue, A b , A d ). A a and A c show the streptavidin labeling only, while A b and A d show the triple labeling. B. High magnification of a biotinylated axon, showing patches of biotinylation along the axon (green fluorescence), and a glass microbead (square) attached to a large patch. The bead is better seen in the inset.

    Article Snippet: Streptavidin-coated beads prepared as in ( ) can be replaced by a commercial version: ProActive® Microspheres, Streptavidin Polymeric: 3 μm diameter, CP01N-1, Bangs Laboratories, Inc., Polysciences Europe GmbH.

    Techniques: Fluorescence, Labeling

    In vivo biotinylation of GTA-tagged protein and removal of endogenously biotinylated proteins by Tobacco etch virus (TEV) cleavage. a Western blot detection of biotinylated proteins in lysed wild-type N2 animals ( left lane ) and lysed animals expressing GTA-tagged DLG-1 and ubiquitously expressed BirA ( right lane ). An additional band of the correct estimated molecular weight for DLG-1::GTA is detected upon expression of BirA ( arrow ). b Western blot analysis of eluate and beads after purification and release of bound bait protein by TEV cleavage. Both blots contain the same samples. The background of naturally biotinylated proteins remains bound to the streptavidin beads and is visible on the α-biotin western blot in the beads remainder, while tagged DLG-1 is cleaved off and visible on the α-GFP western blot in the eluate. No DLG-1 protein is purified in samples from animals not expressing BirA. c Lanes 1 and 2 : Western blot analysis of the levels of GTA::CDC-42 purified with GFP-Trap beads ( lane 1 ) and streptavidin beads ( lane 2 ) from animals expressing BirA in seam and hyp7 epidermal cells. Lanes 3 and 4 : Western blot analysis of beads ( lane 3 ) and eluate ( lane 4 ) after cleavage of GTA::CDC-42 purified with streptavidin beads. d Schematic of the TEV cleavage procedure

    Journal: BMC Biology

    Article Title: A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large

    doi: 10.1186/s12915-016-0286-x

    Figure Lengend Snippet: In vivo biotinylation of GTA-tagged protein and removal of endogenously biotinylated proteins by Tobacco etch virus (TEV) cleavage. a Western blot detection of biotinylated proteins in lysed wild-type N2 animals ( left lane ) and lysed animals expressing GTA-tagged DLG-1 and ubiquitously expressed BirA ( right lane ). An additional band of the correct estimated molecular weight for DLG-1::GTA is detected upon expression of BirA ( arrow ). b Western blot analysis of eluate and beads after purification and release of bound bait protein by TEV cleavage. Both blots contain the same samples. The background of naturally biotinylated proteins remains bound to the streptavidin beads and is visible on the α-biotin western blot in the beads remainder, while tagged DLG-1 is cleaved off and visible on the α-GFP western blot in the eluate. No DLG-1 protein is purified in samples from animals not expressing BirA. c Lanes 1 and 2 : Western blot analysis of the levels of GTA::CDC-42 purified with GFP-Trap beads ( lane 1 ) and streptavidin beads ( lane 2 ) from animals expressing BirA in seam and hyp7 epidermal cells. Lanes 3 and 4 : Western blot analysis of beads ( lane 3 ) and eluate ( lane 4 ) after cleavage of GTA::CDC-42 purified with streptavidin beads. d Schematic of the TEV cleavage procedure

    Article Snippet: Affinity purification Affinity purifications were performed with streptavidin-coated beads (Chromotek, HP57.1).

    Techniques: In Vivo, Western Blot, Expressing, Molecular Weight, Purification

    In vivo biotinylation is highly tissue-specific. a Expression of GFP::TEV::Avi from the intestinal elt-2 promoter. b Expression of GFP::TEV::Avi from the seam and hyp7 epidermal cell-specific wrt-2 promoter. In a and b, the tissue and approximate region imaged are indicated in the cartoon worm. c Western blot detection of GFP::TEV::Avi purified with streptavidin beads from lysates obtained from animals expressing GFP::TEV::Avi and BirA in the same tissue (lanes 1 and 3) or in different tissues (lanes 2 and 4). Scale bars are 10 μm

    Journal: BMC Biology

    Article Title: A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large

    doi: 10.1186/s12915-016-0286-x

    Figure Lengend Snippet: In vivo biotinylation is highly tissue-specific. a Expression of GFP::TEV::Avi from the intestinal elt-2 promoter. b Expression of GFP::TEV::Avi from the seam and hyp7 epidermal cell-specific wrt-2 promoter. In a and b, the tissue and approximate region imaged are indicated in the cartoon worm. c Western blot detection of GFP::TEV::Avi purified with streptavidin beads from lysates obtained from animals expressing GFP::TEV::Avi and BirA in the same tissue (lanes 1 and 3) or in different tissues (lanes 2 and 4). Scale bars are 10 μm

    Article Snippet: Affinity purification Affinity purifications were performed with streptavidin-coated beads (Chromotek, HP57.1).

    Techniques: In Vivo, Expressing, Western Blot, Purification

    DNA hairpin substrate design. Schematic representation of the DNA hairpin substrate extended between two trapped beads (not to scale). The construct consisted of three separate fragments ligated together: a 1550-bp ‘Left handle’ (L; cyan), a 1500-bp ‘Right handle’ (R; dark blue), and the 89-bp (with (dT) 4 tetraloop) hairpin (red). L and R served as linkers attached to the trapped beads via 5′-biotin and 5′-digoxigenin modifications, respectively. L was attached to a 0.79-μm streptavidin-coated bead, R to a 0.86-μm anti-digoxigenin-coated bead. The hairpin was flanked at the 5′ end by a (dT) n ssDNA region that serves as a helicase loading site (here n = 10) and at the 3′ end by an abasic site (dark blue open circle). The construct was modular, allowing the hairpin sequence and the loading site length to be varied (see ‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.004

    Journal: eLife

    Article Title: Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

    doi: 10.7554/eLife.00334

    Figure Lengend Snippet: DNA hairpin substrate design. Schematic representation of the DNA hairpin substrate extended between two trapped beads (not to scale). The construct consisted of three separate fragments ligated together: a 1550-bp ‘Left handle’ (L; cyan), a 1500-bp ‘Right handle’ (R; dark blue), and the 89-bp (with (dT) 4 tetraloop) hairpin (red). L and R served as linkers attached to the trapped beads via 5′-biotin and 5′-digoxigenin modifications, respectively. L was attached to a 0.79-μm streptavidin-coated bead, R to a 0.86-μm anti-digoxigenin-coated bead. The hairpin was flanked at the 5′ end by a (dT) n ssDNA region that serves as a helicase loading site (here n = 10) and at the 3′ end by an abasic site (dark blue open circle). The construct was modular, allowing the hairpin sequence and the loading site length to be varied (see ‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.004

    Article Snippet: The hairpin constructs were functionalized with 5′ biotin and 5′ digoxigenin ends, and were tethered between a trapped streptavidin-coated bead (0.79 μm; Spherotech Inc., Lake Forest, IL) and an anti-digoxigenin-coated protein G bead (0.86 μm; Spherotech Inc., Lake Forest, IL) in the laminar flow cell.

    Techniques: Construct, Sequencing

    Laminar flow cell design. Photograph and schematic of a typical experimental flow cell. The flow cell consisted of two microscope cover glasses sandwiching one piece of patterned parafilm (see ‘Materials and methods’). Eight small inlet holes on the top cover glass (four on each side) allowed different buffers to be streamed in through tubing mounted into the chamber bracket. In this photograph, food dye of different colors was flowed into the cell at a rate of 100 μl/hr to illustrate the four streams. The two inner streams (red and blue) merge into a central channel at a point near the center of the flow cell. Due to the laminar flow, a sharp interface is maintained between the two streams. The zoom-in shows the corresponding schematic of the experimental flow cell. The top channel (yellow) was loaded with streptavidin beads with attached DNA substrate. The bottom channel (green) was loaded with anti-DIG beads. The two types of beads diffused into the central channel via thin glass capillaries inserted into the parafilm, where they could be trapped and manipulated. In a typical measurement of single-XPD helicase activity, the top stream (blue) contained ATP while the bottom stream (red) contained protein (‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.006

    Journal: eLife

    Article Title: Sequence-dependent base pair stepping dynamics in XPD helicase unwinding

    doi: 10.7554/eLife.00334

    Figure Lengend Snippet: Laminar flow cell design. Photograph and schematic of a typical experimental flow cell. The flow cell consisted of two microscope cover glasses sandwiching one piece of patterned parafilm (see ‘Materials and methods’). Eight small inlet holes on the top cover glass (four on each side) allowed different buffers to be streamed in through tubing mounted into the chamber bracket. In this photograph, food dye of different colors was flowed into the cell at a rate of 100 μl/hr to illustrate the four streams. The two inner streams (red and blue) merge into a central channel at a point near the center of the flow cell. Due to the laminar flow, a sharp interface is maintained between the two streams. The zoom-in shows the corresponding schematic of the experimental flow cell. The top channel (yellow) was loaded with streptavidin beads with attached DNA substrate. The bottom channel (green) was loaded with anti-DIG beads. The two types of beads diffused into the central channel via thin glass capillaries inserted into the parafilm, where they could be trapped and manipulated. In a typical measurement of single-XPD helicase activity, the top stream (blue) contained ATP while the bottom stream (red) contained protein (‘Materials and methods’). DOI: http://dx.doi.org/10.7554/eLife.00334.006

    Article Snippet: The hairpin constructs were functionalized with 5′ biotin and 5′ digoxigenin ends, and were tethered between a trapped streptavidin-coated bead (0.79 μm; Spherotech Inc., Lake Forest, IL) and an anti-digoxigenin-coated protein G bead (0.86 μm; Spherotech Inc., Lake Forest, IL) in the laminar flow cell.

    Techniques: Flow Cytometry, Microscopy, Activity Assay

    CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. (A) Cell lysates of LPS-primed BMDMs were incubated with different concentrations of biotin–CY-09, which were then pulled down with streptavidin beads. (B) Cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells were incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (C) Purified human NLRP3 protein was incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) Purified human NLRP3 protein was incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (E) MST assay for the affinity between CY-09 and purified GFP-NLRP3 protein. (F and G) Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, AIM2, NLRC4, NLRP3–LRR, NLPR3–NACHT, or NLRP3–PYD constructs were incubated with indicated concentration of biotin–CY-09, which were then pulled down with streptavidin beads. Data are representative of three independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

    doi: 10.1084/jem.20171419

    Figure Lengend Snippet: CY-09 binds to the ATP-binding site of NLRP3 NACHT domain. (A) Cell lysates of LPS-primed BMDMs were incubated with different concentrations of biotin–CY-09, which were then pulled down with streptavidin beads. (B) Cell lysates of LPS-primed BMDMs or PMA-differentiated THP-1 cells were incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (C) Purified human NLRP3 protein was incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) Purified human NLRP3 protein was incubated with biotin–CY-09 and different concentrations of free CY-09, which were then pulled down with streptavidin beads. (E) MST assay for the affinity between CY-09 and purified GFP-NLRP3 protein. (F and G) Cell lysates from HEK-293T cells transfected with Flag-tagged NLRP3, NOD1, NOD2, AIM2, NLRC4, NLRP3–LRR, NLPR3–NACHT, or NLRP3–PYD constructs were incubated with indicated concentration of biotin–CY-09, which were then pulled down with streptavidin beads. Data are representative of three independent experiments.

    Article Snippet: Protein G agarose and streptavidin-coated beads were respectively supplied by Millipore and Pierce Biochemicals.

    Techniques: Binding Assay, Incubation, Purification, Microscale Thermophoresis, Transfection, Construct, Concentration Assay

    CY-09 inhibits NLRP3 ATPase activity. (A) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CY-09. (B) ATPase activity assay for purified Flag–NLRP3, NLRC4, NLRP1, NOD2 or RIG-I with or without presence of CY-09 (1 µM). (C) Cell lysates from HEK-293T cells transfected with Flag-tagged WT NLRP3 or NLRP3 constructs with Walker A or Walker B motif mutation were incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) ATP-binding assay for purified Flag–NLRP3 in the presence of different concentrations of CY-09. (E) ATPase activity assay for purified Flag–NLRP3 or mutants with or without presence of CY-09 (1 µM). (F) Docking complex of NLRP3 with CY-09. CY-09 is shown in sticks and colored green, while NLRP3 is shown in cartoon and colored light blue. Data are from three independent experiments with biological duplicates in each (A, B, and E; mean and SEM of n = 6) or are representative of two or three independent experiments (C and D). Statistics were analyzed using an unpaired Student’s t test: ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Identification of a selective and direct NLRP3 inhibitor to treat inflammatory disorders

    doi: 10.1084/jem.20171419

    Figure Lengend Snippet: CY-09 inhibits NLRP3 ATPase activity. (A) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CY-09. (B) ATPase activity assay for purified Flag–NLRP3, NLRC4, NLRP1, NOD2 or RIG-I with or without presence of CY-09 (1 µM). (C) Cell lysates from HEK-293T cells transfected with Flag-tagged WT NLRP3 or NLRP3 constructs with Walker A or Walker B motif mutation were incubated with different concentrations of biotin–CY-09 and then pulled down with streptavidin beads. (D) ATP-binding assay for purified Flag–NLRP3 in the presence of different concentrations of CY-09. (E) ATPase activity assay for purified Flag–NLRP3 or mutants with or without presence of CY-09 (1 µM). (F) Docking complex of NLRP3 with CY-09. CY-09 is shown in sticks and colored green, while NLRP3 is shown in cartoon and colored light blue. Data are from three independent experiments with biological duplicates in each (A, B, and E; mean and SEM of n = 6) or are representative of two or three independent experiments (C and D). Statistics were analyzed using an unpaired Student’s t test: ***, P

    Article Snippet: Protein G agarose and streptavidin-coated beads were respectively supplied by Millipore and Pierce Biochemicals.

    Techniques: Activity Assay, Purification, Transfection, Construct, Mutagenesis, Incubation, Binding Assay

    AdoHcy binds the DENV3 MTase with a much weaker affinity than do AdoMet and SIN. ( A ) Dose response of inhibition of the [ 3 H]-SAM-MTase complex formation by AdoMet (black), AdoHcy (red), and SIN (green). The biotinylated DENV3 MTase and 3 H-labeled SAM were incubated with or without compounds AdoMet, AdoHcy, and SIN. A two-fold dilution series was shown for each compound. The reaction mixtures were mixed with the streptavidin-coated SPA beads and quantified using a Microbeta 2 scintillation counter. ( B ). Superposition of the crystal structures of the MTase-SIN complex (green) [ 23 ] and the MTase-SAH complex (yellow) [ 19 ]. SAH and SIN are shown in stick representation. Atomic color coding is as follows (unless otherwise specified): carbon in yellow/green, oxygen in red, nitrogen in blue, and sulfur in orange. Potential hydrogen bonds are depicted in red dashed lines.

    Journal: PLoS ONE

    Article Title: S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

    doi: 10.1371/journal.pone.0076900

    Figure Lengend Snippet: AdoHcy binds the DENV3 MTase with a much weaker affinity than do AdoMet and SIN. ( A ) Dose response of inhibition of the [ 3 H]-SAM-MTase complex formation by AdoMet (black), AdoHcy (red), and SIN (green). The biotinylated DENV3 MTase and 3 H-labeled SAM were incubated with or without compounds AdoMet, AdoHcy, and SIN. A two-fold dilution series was shown for each compound. The reaction mixtures were mixed with the streptavidin-coated SPA beads and quantified using a Microbeta 2 scintillation counter. ( B ). Superposition of the crystal structures of the MTase-SIN complex (green) [ 23 ] and the MTase-SAH complex (yellow) [ 19 ]. SAH and SIN are shown in stick representation. Atomic color coding is as follows (unless otherwise specified): carbon in yellow/green, oxygen in red, nitrogen in blue, and sulfur in orange. Potential hydrogen bonds are depicted in red dashed lines.

    Article Snippet: We therefore mixed the biotinylated DENV3 MTase with streptavidin-coated SPA beads (PerkinElmer).

    Techniques: Inhibition, Labeling, Incubation