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  • 99
    Vector Laboratories streptavidin
    Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a <t>streptavidin-coated</t> coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003
    Streptavidin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher strepavidin antibody
    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through <t>biotin–streptavidin</t> interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.
    Strepavidin Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega streptavidin
    (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled <t>streptavidin</t> or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.
    Streptavidin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend streptavidin
    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and <t>streptavidin-APC.</t> Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.
    Streptavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend pe strepavidin
    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and <t>streptavidin-APC.</t> Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.
    Pe Strepavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    BioLegend streptavidin apc
    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and <t>streptavidin-APC.</t> Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.
    Streptavidin Apc, supplied by BioLegend, used in various techniques. Bioz Stars score: 98/100, based on 603 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    BioLegend hrp streptavidin
    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and <t>streptavidin-APC.</t> Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.
    Hrp Streptavidin, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam strepavidin hrp
    SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by <t>IgG-HRP</t> or (B) <t>Streptavidin-HRP.</t> VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =
    Strepavidin Hrp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher strepavidin hrp
    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or <t>HRP-conjugated</t> <t>streptavidin.</t> (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).
    Strepavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher streptavidin pe
    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a <t>streptavidin</t> peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.
    Streptavidin Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore atto550 strepavidin
    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a <t>streptavidin</t> peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.
    Atto550 Strepavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    BioLegend strepavidin fitc
    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a <t>streptavidin</t> peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.
    Strepavidin Fitc, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin pe cy7
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Streptavidin Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Innovex Biosciences strepavidin
    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and <t>streptavidin-conjugated</t> microbeads. Sca-1 + cells were enriched in a second MACS step with <t>Sca-1-PE-Cy7</t> antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p
    Strepavidin, supplied by Innovex Biosciences, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biacore streptavidin
    Kinetics of inhibition of biotinylated D336C channels by <t>streptavidin.</t> (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via <t>biotin–streptavidin–biotin</t> interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.
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    Image Search Results


    Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a streptavidin-coated coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003

    Journal: eLife

    Article Title: Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains

    doi: 10.7554/eLife.00943

    Figure Lengend Snippet: Dynein crosslinks and slides MTs in bundles. ( A ) Schematic overview of the dynein constructs used in this study. The N-terminal tail is shown in gray, the linker in purple, the six numbered AAA+ domains are in light blue and the stalk and MT binding domain are depicted in orange. GFP and GST tags are shown in green and blue, respectively. The Halo tag (DHA, Promega) is shown in red. ( B ) Coomassie brilliant blue stained gels showing purified dynein constructs used in this study. The associated subunits of the brain cytoplasmic dynein complex are labeled; HC–heavy chain, IC–intermediate chain, LIC–light intermediate chain, LC–light chain. Recombinant yeast dynein constructs do not contain associated subunits. Molecular weight markers are indicated. ( C ) MTs incubated in the absence or presence of dynein are visualized by attachment to a streptavidin-coated coverslip via a biotin tag. Brain dynein and GST-Dyn1 331kDa crosslink MTs into large bundles, while the dynein monomer, Dyn1 331kDa does not. Scale bar, 10 µm. ( D ) Cartoon depicting two different mechanisms by which dynein could crosslink MTs, either using its two motor domains or through the tail domain. Alexa-568 and Alexa-488 labeled MTs are crosslinked by dynein. The green MTs are attached to the coverslip through a biotin-streptavidin linkage and perfusion of 1 mM ATP induces sliding between the MTs. ( E and F ) Example of rat ( E ) and GST-Dyn1 331kDa ( F ) dynein-driven sliding of red-labeled MTs within the bundle after 1 mM ATP addition. Arrowhead tracks the sliding MT within the bundle. The time relative to the start is noted in min:s at the bottom of each image. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.00943.003

    Article Snippet: The chamber was coated sequentially with the following solutions: 5 mg/ml BSA-biotin (Sigma, St. Louis, MO), 60 µl BC buffer (BRB80, 1 mg/ml BSA, 1 mg/ml casein, 0.5% Pluronic F-168, pH 6.8), 20 µl 0.5 mg/ml streptavidin (Vector Labs, Burlingame, CA), and 60 µl BC buffer to remove excess streptavidin.

    Techniques: Construct, Binding Assay, Staining, Purification, Labeling, Recombinant, Molecular Weight, Incubation

    The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Journal: Nucleic Acids Research

    Article Title: A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

    doi: 10.1093/nar/gkv069

    Figure Lengend Snippet: The concept of a high-throughput screening method to identify compounds or proteins targeting protein–nucleic acids interactions. The first step is to link a biotin-labeled oligomer to surface of a multiple-well plate through biotin–streptavidin interaction. After the surface-blocking step, a nucleic acids–binding protein is added to the wells to bind to the oligomer. The compounds or proteins of interest can also be added to the wells to inhibit or enhance the protein binding, which is the basis of the method for high-throughput drug screening. Colorimetric, chemiluminescence or fluorescence methods can be used to detect whether the compounds or proteins inhibit or enhance the binding capacities of the nucleic acids–binding protein.

    Article Snippet: Materials Biotin-labeled hairpin DNA oligomer FL814 containing a specific binding site of HMGA2 was purchased from Eurofins MWG Operon, Inc. Streptavidin covalently coated 96-well plates (NUNC Immobilizer Streptavidin-F96 clear) were from Thermo Fisher Scientific, Inc. Antibody against HMGA2 (HMGA2 (D1A7) Rabbit mAb) and Anti-rabbit IgG, HRP-linked Antibody #7074 were purchased from Cell Signaling, Inc. Ultra TMB-ELISA was bought from Thermo Fisher Scientific, Inc.

    Techniques: High Throughput Screening Assay, Labeling, Blocking Assay, Binding Assay, Protein Binding, Fluorescence

    (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled streptavidin or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.

    Journal: ACS applied materials & interfaces

    Article Title: Fabrication of Oligonucleotide and Protein Arrays on Rigid and Flexible Substrates Coated with Reactive Polymer Multilayers

    doi: 10.1021/am302285n

    Figure Lengend Snippet: (A–B) Representative fluorescence micrographs of glass substrates coated with PEI/PVDMA films spotted with (A) FITC-labeled streptavidin or (B) unlabeled streptavidin (the image in (B) was treated with anti-HA-biotin and anti-rat IgG Alexa Fluor 488 prior to imaging; see text). (C) Digital photograph of film-coated glass containing spots of immobilized β-galactosidase (right spot) or BSA (left spot) incubated under droplets of ONPG for 10 minutes. (D) Plot of ONP concentration vs. time measured from a droplet incubated on a spot containing immobilized β-galactosidase (black squares) or BSA (grey diamonds). Scale bars are (A–B) 1 mm and (C) 2 mm.

    Article Snippet: Streptavidin was purchased from Promega (Madison, WI). β-Galactosidase (β-Gal) was purchased from Prozyme (Hayward, CA).

    Techniques: Fluorescence, Labeling, Imaging, Incubation, Concentration Assay

    Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and streptavidin-APC. Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Galectin-9 controls the therapeutic activity of 4-1BB–targeting antibodies

    doi: 10.1084/jem.20132687

    Figure Lengend Snippet: Defective 4-1BB activity in T cells and DCs from Gal-9 −/− mice. (a) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD8 + T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (b) 4-1BB and OX40 surface expression on WT and Gal-9 −/− CD8 + T cells activated as in panel a. Mean fluorescent intensity indicated for 4-1BB. Shade, isotype control. (c) Binding of anti–4-1BB (clone 3H3) to activated CD8 + T cells. Activated CD8 + T cells as in panel a were sorted for identical expression of 4-1BB using biotin-anti–4-1BB (Syrian hamster IgG; clone 17B5) and streptavidin-APC. Cells were then stained with unlabeled anti–4-1BB (rat IgG; clone 3H3) and anti–rat IgG FITC to detect anti–4-1BB (clone 3H3) binding to cells. Shade, isotype controls. Data are representative of two different experiments. (d) IFN-γ production by preactivated CD8 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (postsort FACS plot shown) and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of at least three different experiments. (e and f) CD8 + T cells from WT or Gal-9 −/− mice were activated as in panel a, sorted for identical expression of 4-1BB, and recultured for another 24 h to assay IFN-γ production in response to irradiated 4-1BBL + hybridoma cells (e) or varying concentrations of anti-polyhistidine cross-linked r4-1BBL (f). Neutralizing anti–4-1BBL or rat IgG was added to cultures in e. Data are means ± SEM from triplicate cultures and representative of two different experiments. (g, left) Surface expression of 4-1BB (top) and Gal-9 (bottom) in splenic CD4 T cells activated in vitro with anti-CD3 and anti-CD28 for 72 h. Shade, isotype control. (right) IL-2 production by preactivated CD4 + T cells, from WT or Gal-9 −/− mice, sorted for identical expression of 4-1BB (pre- and postsort FACS plot shown), and restimulated with plate-bound anti-CD3 in the presence of control rat IgG or anti–4-1BB for 24 h. Data are means ± SEM from triplicate cultures and representative of three different experiments. (h) Surface expression of 4-1BB and Gal-9 (top) and 4-1BB and MHC II (bottom) on ex vivo WT mesenteric LN DCs and splenic DCs from WT and Gal-9 −/− mice cultured with GM-CSF, respectively. Shade, isotype control. (i) ALDEFLUOR staining in preactivated spleen DCs from WT and Gal-9 −/− mice, sorted for identical expression of 4-1BB, and recultured for 24 h in the presence of zymosan with control rat IgG (left) or agonist anti–4-1BB (right). Numbers indicate percentage of cells in each quadrant. Data are representative of three different experiments.

    Article Snippet: The following antibodies were used for flow cytometry: PE-conjugated anti–Gal-9 (108A2) and anti-CD25 (PC61), PE-Cy7–conjugated anti-NK1.1 (PK136) and anti–IFN-γ (XMG1.2), FITC-conjugated anti-CD44 and anti-CD49b (DX5), peridinin chlorophyll protein–conjugated CD62L (MEL-14), peridinin chlorophyll protein–Cy5.5-conjugated CD8β and anti–L-17A, Pacific blue–conjugated anti-CD3ε (145-2C11) and anti-CD4 (GK1.5), biotin-conjugated anti–4-1BB and anti-OX40, allophycocyanin-conjugated anti–Gr-1 (RB6-8C5), anti–rat IgG (Poly4054), and streptavidin (all from BioLegend); PE-conjugated anti–4-1BBL (TKS-1) and anti–Gal-3 (M3/38), Alexa Flour 700–conjugated MHC class II (M5/114.15.2), and FITC-conjugated anti-CD11c (N418; all from eBioscience).

    Techniques: Activity Assay, Mouse Assay, Expressing, In Vitro, Binding Assay, Staining, FACS, Irradiation, Ex Vivo, Cell Culture

    SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by IgG-HRP or (B) Streptavidin-HRP. VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =

    Journal: Frontiers in Microbiology

    Article Title: A Periplasmic Antimicrobial Peptide-Binding Protein Is Required for Stress Survival in Vibrio cholerae

    doi: 10.3389/fmicb.2019.00161

    Figure Lengend Snippet: SipA binds polymyxin B and LL-37 in vitro and in vivo . Wells from a 96-well plate were coated with either purified SipA or VC1638 (A,B) . LL-37 (A) or biotin-labeled polymyxin B (B) were added to plates and incubated for 1 h. Levels of binding were detected via (A) anti-LL-37 antibody followed by IgG-HRP or (B) Streptavidin-HRP. VC1638 served as a control protein. (C) Coimmunoprecipitation of LL-37 in the presence and absence of 6×His- sipA . Each culture was crosslinked and lysed before loading onto a Ni-NTA column (input), followed by elution with imidazole (output). Blots were probed with anti-LL-37 antibody. Densitometry of the LL-37 band was calculated based on three separate experiments and normalized to the strain containing empty vector. ∗ p =

    Article Snippet: Plates were washed and either LL-37 or biotinylated polymyxin B were added at 10 μg/mL and incubated for 1 h. Plates were washed again and anti-LL-37 (mouse; Santa-Cruz Biotechnology), was added for 1 h. After washing, either Goat pAb to Mouse IgG + IgM HRP (Abcam), or Strepavidin-HRP (Abcam) was added.

    Techniques: In Vitro, In Vivo, Purification, Labeling, Incubation, Binding Assay, Plasmid Preparation

    Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Journal: Molecular Biology of the Cell

    Article Title: Dynamics of human telomerase recruitment depend on template-telomere base pairing

    doi: 10.1091/mbc.E17-11-0637

    Figure Lengend Snippet: Imetelstat (GRN163L) is a competitive inhibitor of primer-substrate binding by telomerase. (A) Experimental design of single-molecule telomerase primer binding and activity assay. Halo-telomerase is modified with a biotin-HaloTag-ligand and immobilized on the coverslip surface using NeutrAvidin. Primer binding is visualized by telomerase-dependent recruitment of a fluorescent primer to the coverslip surface. The telomerase extension product is detected using a fluorescently labeled oligonucleotide anti-sense to the telomerase extension product. (B) Western blot and fluorescence imaging of Halo-telomerase modified with a fluorescent dye (JF646) or biotin, probed with an anti-TERT antibody or HRP-conjugated streptavidin. (C) Single-molecule TIRF imaging of primer molecules recruited to the coverslip surface by telomerase (top) and its colocalization with telomerase extension products after incubation with nucleotide substrate (bottom). (D) Single-molecule TIRF imaging of primer binding by telomerase in the presence of increasing concentrations of imetelstat. (E) Quantification of primer binding to telomerase as a function of imetelstat concentration ( n = 5 fields of view per concentration, data points plotted as mean ± SD, error on IC 50 reflects error in the corresponding fit of the data to a simple binding curve). (F) Direct telomerase assay at 150 mM KCl in the absence and presence of imetelstat (10 nM), or mismatched control oligonucleotide (MM Control, 10 nM), and increasing concentrations of primer substrate. LC1, LC2, and LC3, labeled DNA loading controls. (G) Quantification of telomerase activity as a function of primer concentration in absence and presence of imetelstat (10 nM) or mismatched control oligonucleotide (MM Control, 10 nM).

    Article Snippet: The HaloTag modified with biotin was detected using Strepavidin-HRP (Pierce; 1:2000).

    Techniques: Binding Assay, Activity Assay, Modification, Labeling, Western Blot, Fluorescence, Imaging, Incubation, Concentration Assay, Telomerase Assay

    Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a streptavidin peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.

    Journal: The Journal of Experimental Medicine

    Article Title: Presentation of Antigen in Immune Complexes Is Boosted by Soluble Bacterial Immunoglobulin Binding Proteins

    doi:

    Figure Lengend Snippet: Effect of two SpA derivatives on the binding to coated hIgMs and on the T cell presentation of toxin α. (A) Mα2-3 was incubated overnight in the presence or absence of fixed concentrations of toxin α biotinylated at the NH 2 terminus (Alphabiot) and either ZZ or BB in BSA-coated microwell plates. The solutions were transferred in hIgM-coated plates. Binding of Mα2-3 to the wells was determined using a goat anti–mouse IgG peroxidase conjugate (GAM–PO), whereas binding of biotinylated toxin α was determined using a streptavidin peroxidase conjugate (SA–PO). (B) Toxin α (alpha) was serially diluted and incubated overnight at 4°C in the presence or absence of mAb Mα2-3 (25 nM final) and either ZZ or BB (0.1 μM final for each derivative). 5 × 10 5 splenocytes from BALB/c mice were then added to each well in the presence of 5 × 10 4 T1B2. T cell stimulation was assessed as previously described.

    Article Snippet: Streptavidin-PE (SAPE) was purchased from Caltag Labs.

    Techniques: Binding Assay, Incubation, Mouse Assay, Cell Stimulation

    A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1 + cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p

    Journal: Frontiers in Endocrinology

    Article Title: Transcriptional Pathways in cPGI2-Induced Adipocyte Progenitor Activation for Browning

    doi: 10.3389/fendo.2015.00129

    Figure Lengend Snippet: A MACS selection procedure for the prospective isolation of progenitor cells for beige/brite differentiation . (A–F) Single cell suspensions were obtained by collagenase digestion of subcutaneous fat, and stained for six-color FACS with the indicated antibodies after removal of adipocytes by centrifugation and erythrocytes by TER119-MACS ® depletion. Debris and singlets were excluded and selected through FSC/SSC and FSC-A/H, respectively. (A–C) and (D–F) represent independent gating schemes. Values (%) indicate cells % of parent plot. Representative plots from multiple independent experiments are shown. (G) Erythrocytes, leukocytes, and endothelial cells were removed from single cell suspensions in a first MACS step with biotinylated Ter119, CD45, and CD31 antibodies and streptavidin-conjugated microbeads. Sca-1 + cells were enriched in a second MACS step with Sca-1-PE-Cy7 antibody and anti-PE-Cy7 microbeads. The resulting cell population (Lin − Sca-1 + eluate) as well as the Lin − flow-through were subjected to flow cytometry. Comparable purities were obtained with directly conjugated antibody-bead combinations (data not shown). (H) MACS- and FACS-purified cells were cultured and differentiated for 8 days in the presence or absence of cPGI 2 (see Materials and Methods ) before subjected to RNA expression analysis by qRT-PCR (* indicates t -test p

    Article Snippet: The flow-through was collected and stained with CD45-FITC (30-F11, eBioscience), CD31-eFluor 450 (390, eBioscience), CD29-PerCP-eFluor 710 (HMb1-1, eBioscience), CD34-Alexa Fluor 647 (RAM34, BD Biosciences, Heidelberg, Germany), Sca-1-Alexa Fluor 700 (D7, eBioscience), and CD140a(Pdgfrα)-biotin (APA5, eBioscience) for 30 min on ice, followed by staining with streptavidin-PE-Cy7 (eBioscience).

    Techniques: Magnetic Cell Separation, Selection, Isolation, Staining, FACS, Centrifugation, Flow Cytometry, Cytometry, Purification, Cell Culture, RNA Expression, Quantitative RT-PCR

    Kinetics of inhibition of biotinylated D336C channels by streptavidin. (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.

    Journal: The Journal of General Physiology

    Article Title: Constraints on Voltage Sensor Movement in the Shaker K+ Channel

    doi: 10.1085/jgp.200609624

    Figure Lengend Snippet: Kinetics of inhibition of biotinylated D336C channels by streptavidin. (A) Pulse protocols are as described in Fig. 4 , except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol ( n = 6), circles; long-pulse protocol ( n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.

    Article Snippet: Capture rates of streptavidin to biotin-conjugated BSA immobilized on a BIAcor chip, a lipid-free system, are even faster, displaying association rate constants two orders of magnitude greater than in the bilayer system , suggesting that partitioning of biotin into the lipid may affect its aqueous accessibility in the more biological systems.

    Techniques: Inhibition, Mass Spectrometry, Generated

    Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via biotin–streptavidin–biotin interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.

    Journal: APL Bioengineering

    Article Title: Nanoerythrosome-functionalized biohybrid microswimmers

    doi: 10.1063/1.5130670

    Figure Lengend Snippet: Biohybrid bacterial microswimmers functionalized with red blood cell (RBC)-membrane nanoliposomes (nanoerythrosomes). (a) Biohybrid bacterial microswimmers are composed of genetically engineered motile E. coli MG1655 substrain and nanoerythrosomes made out of the RBC membranes. Nanoerythrosomes are functionalized on the bacterial membrane via biotin–streptavidin–biotin interaction. The nanoerythrosome membrane is biotinylated by functionalization of TER-119 antibodies with biotin molecules, whereas E. coli MG1655 is bioengineered to express biotin attachment peptides on its membrane and biotin molecules are directly conjugated on its membrane surface. (b) Scanning electron microscopy (SEM) image of the nanoerythrosome functionalized bacterial swimmers. Nanoerythrosomes were conjugated not only to the bacterial body but also to their flagella. (c) A typical swimming trajectory for the biohybrid bacterial microswimmers captured via fluorescent microscopy. The red dot indicates nanoerythrosome on bacteria and the white line shows the swimming trajectory. The inset shows bacteria (green color) functionalized with nanoerythrosomes (red color). The scale bar of the inset is 1 μ m.

    Article Snippet: Briefly, after the bacteria culture reached 0.6 OD600 , 1 ml bacteria culture was rinsed two times with motility buffer [10 mM K2 HPO4 , 10 mM KHPO4 , 67 mM NaCl, 0.1 mM EDTA and 1% (w/v) glucose, pH 7] at RT and 2000g and then incubated in motility buffer that contained 100 μ g/ml streptavidin (Sigma-Aldrich, St. Louis, MO) at 37 °C and 200 rpm for 1 h. After the incubation period, the sample was rinsed two times at RT and 2000g to remove the unbounded streptavidin molecules and then resuspended in 1 ml motility buffer to use in the conjugation procedure.

    Techniques: Electron Microscopy, Microscopy