r-phycoerythrin-streptavidin Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Vector Laboratories streptavidin r phycoerythrin
    Streptavidin R Phycoerythrin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin/product/Vector Laboratories
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher r phycoerythrin labeled streptavidin
    Schematic of BEAMing. Step 1: Magnetic beads covalently coated with <t>streptavidin</t> are bound to biotinylated oligonucleotides (oligos). Step 2: An aqueous mix containing all the necessary components for PCR plus primer-bound beads and template DNA are stirred together with an oil/detergent mix to create microemulsions. The aqueous compartments (white circles in the gray oil layer) contain an average of less than one template molecule and less than one bead. Red and green templates represent two template molecules, the sequences of which differ by one or many nucleotides. Step 3: The microemulsions are temperature-cycled as in a conventional PCR. If a DNA template and a bead are present together in a single aqueous compartment, the bead-bound oligonucleotides act as primers for amplification. The straight red and green lines connected to the beads represent extension products from the two different kinds of templates. Step 4: The emulsions are broken, and the beads are purified with a magnet. Step 5: After denaturation, the beads are incubated with oligonucleotides that can distinguish between the sequences of the different kinds of templates. Fluorescently labeled antibodies then are used to label the bound hybridization probes, which renders the beads containing PCR product as red or green after appropriate laser excitation. Step 6: Flow cytometry is used to count the red and green beads.
    R Phycoerythrin Labeled Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r phycoerythrin labeled streptavidin/product/Thermo Fisher
    Average 99 stars, based on 156 article reviews
    Price from $9.99 to $1999.99
    r phycoerythrin labeled streptavidin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    89
    Thermo Fisher streptavidin r pe
    A representative FACS analysis of ocular infiltrating cells before v after PC. A flow cytometric analysis was carried out on each ocular infiltration inflammatory cells. Left panel: The discrimination of ocular infiltrating cells by forward and side scattered gate. Only viable cells were analysed in the live gate. Typical neutrophil and macrophage gate and NK cell and lymphocyte gate were indicated. Right panel: Neutrophil and macrophage: The rectangular gates indicated neutrophils and macrophages. Neutrophils were stained with FITC conjugated anti-Gr-1 mAb and Cy-chrome conjugated anti-CD45 mAb. Macrophages were stained with biotin conjugated anti-F4/80 monoclonal antibody (mAb) counterstained by <t>Streptavidin/R-PE</t> and Cy-chrome conjugated anti-CD45 mAb. Lymphocyte: The quadrant gate indicated T cells and B cells. Cells were stained with Cy-chrome conjugated anti-mouse TCR β chain mAb (T cells) and FITC conjugated anti-mouse CD19 mAb (B cells). NK cell: NK cells were indicated by the histogram. Cells were stained with and biotin conjugated anti-mouse NK1. 1 mAb counter stained by Streptavidin/R-PE.
    Streptavidin R Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r pe/product/Thermo Fisher
    Average 89 stars, based on 199 article reviews
    Price from $9.99 to $1999.99
    streptavidin r pe - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    85
    Thermo Fisher streptavidin r pe conjugate
    Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5  Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.
    Streptavidin R Pe Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r pe conjugate/product/Thermo Fisher
    Average 85 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    streptavidin r pe conjugate - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    88
    ProZyme strepavidin r phycoerythrin
    Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5  Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.
    Strepavidin R Phycoerythrin, supplied by ProZyme, used in various techniques. Bioz Stars score: 88/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strepavidin r phycoerythrin/product/ProZyme
    Average 88 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    strepavidin r phycoerythrin - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    84
    Thermo Fisher streptavidin alexa fluor 647 r phycoerythrin conjugate alexa fluor 647 r phycoerythrin streptavidin
    Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5  Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.
    Streptavidin Alexa Fluor 647 R Phycoerythrin Conjugate Alexa Fluor 647 R Phycoerythrin Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin alexa fluor 647 r phycoerythrin conjugate alexa fluor 647 r phycoerythrin streptavidin/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin alexa fluor 647 r phycoerythrin conjugate alexa fluor 647 r phycoerythrin streptavidin - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    99
    Millipore streptavidin r phycoerythrin
    Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5  Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.
    Streptavidin R Phycoerythrin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin/product/Millipore
    Average 99 stars, based on 89 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Jackson Immuno r pe conjugated streptavidin
    Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’) 2 generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate as the target cell and peripheral blood mononuclear cells PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cell in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and <t>streptavidin-PE</t> conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1
    R Pe Conjugated Streptavidin, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r pe conjugated streptavidin/product/Jackson Immuno
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    r pe conjugated streptavidin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    90
    ProZyme streptavidin r phycoerythrin conjugate
    Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’) 2 generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate as the target cell and peripheral blood mononuclear cells PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cell in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and <t>streptavidin-PE</t> conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1
    Streptavidin R Phycoerythrin Conjugate, supplied by ProZyme, used in various techniques. Bioz Stars score: 90/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin conjugate/product/ProZyme
    Average 90 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin conjugate - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    96
    Jackson Immuno r phycoerythrin streptavidin
    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated <t>streptavidin.</t> B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
    R Phycoerythrin Streptavidin, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 96/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r phycoerythrin streptavidin/product/Jackson Immuno
    Average 96 stars, based on 64 article reviews
    Price from $9.99 to $1999.99
    r phycoerythrin streptavidin - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    92
    Becton Dickinson streptavidin r phycoerythrin
    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated <t>streptavidin.</t> B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p
    Streptavidin R Phycoerythrin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin/product/Becton Dickinson
    Average 92 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    Qiagen streptavidin r phycoerythrin
    Hybridization of the InflA probe, with and without an increasing number of dInosines, with targets containing increasing numbers of mismatching nucleotides. The sequence of the InflA probe was designed from a conserved region of the matrix 2 gene in segment 7 of subtype H2N3 of the Influenza A virus ( Figure 1 , and Table 1 sequence of H3N2). The number of dInosines included in the probes is indicated in the names Ino3–Ino21. Similarly, the wobbN_21 probe contains 21 N wobbles in the same position as the dInosines in the Ino21 probe. The complementary target of the InflA probe is named the InflA target. These targets have 0–21 pm, as indicated by the names; the 21-gm target has seven groups of three mismatches ( Figure 2 D). All target molecules were biotinylated at their 5′ end. Each sample contained 0.2 nM of one of the synthetic biotinylated single-stranded targets and a mixture containing 2500 microspheres of each of the unique coupled probes tested. In ( A ) and ( B ), the mixture contained the InflA probe and the Ino3, 5, 7, 9 and 21 probes; in ( C ), the mixture contained the InflA probe, the Ino21 probe and the wobbN_21 probe. Hybridization was performed at 45°C (A and C) and 55°C (B and C) in a buffer containing 3 M TMAC for 30 min, followed by 15-min incubation with <t>Streptavidin-R-phycoerythrin.</t> A total of 100 microspheres per type of probe were analyzed in the Luminex 200; the extent of hybridization is indicated as median fluorescent intensity (MFI) on the y-axis. Each experiment contained triplet samples and was repeated two or three times. The sequences of probes and targets are provided in Supplementary Table 1A and B . ( D ) The figure shows the distribution of dInosines (orange/light blue/green boxes) and N wobbles (light grey boxes) in the probes (grey bar), and the mismatching bases (magenta boxes) in the target molecules (black bar), used in Figure 2 A–C.
    Streptavidin R Phycoerythrin, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin/product/Qiagen
    Average 93 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    96
    Millipore streptavidin
    Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B 12 . BtuCD WT denotes the wild-type, BtuCD cys denotes the cysteine mutant and BtuCD EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B 12 to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a <t>biotin-streptavidin</t> link and imaged using TIRF microscopy. d A complex of BtuCD cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg 2+ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c , but with the ATPase impaired mutant BtuCD EQ . No events were observed. f Similar experiment as described in c , but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d – f around 1000 single-molecule fluorescence traces were analysed
    Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 3837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin/product/Millipore
    Average 96 stars, based on 3837 article reviews
    Price from $9.99 to $1999.99
    streptavidin - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    92
    Bio-Rad streptavidin r phycoerythrin
    Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B 12 . BtuCD WT denotes the wild-type, BtuCD cys denotes the cysteine mutant and BtuCD EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B 12 to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a <t>biotin-streptavidin</t> link and imaged using TIRF microscopy. d A complex of BtuCD cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg 2+ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c , but with the ATPase impaired mutant BtuCD EQ . No events were observed. f Similar experiment as described in c , but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d – f around 1000 single-molecule fluorescence traces were analysed
    Streptavidin R Phycoerythrin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin r phycoerythrin/product/Bio-Rad
    Average 92 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    streptavidin r phycoerythrin - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Agilent technologies r phycoerythrin conjugated streptavidin
    Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B 12 . BtuCD WT denotes the wild-type, BtuCD cys denotes the cysteine mutant and BtuCD EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B 12 to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a <t>biotin-streptavidin</t> link and imaged using TIRF microscopy. d A complex of BtuCD cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg 2+ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c , but with the ATPase impaired mutant BtuCD EQ . No events were observed. f Similar experiment as described in c , but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d – f around 1000 single-molecule fluorescence traces were analysed
    R Phycoerythrin Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r phycoerythrin conjugated streptavidin/product/Agilent technologies
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    r phycoerythrin conjugated streptavidin - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    88
    Becton Dickinson r pe conjugated streptavidin
    LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by <t>streptavidin-PE.</t>
    R Pe Conjugated Streptavidin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/r pe conjugated streptavidin/product/Becton Dickinson
    Average 88 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    r pe conjugated streptavidin - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Schematic of BEAMing. Step 1: Magnetic beads covalently coated with streptavidin are bound to biotinylated oligonucleotides (oligos). Step 2: An aqueous mix containing all the necessary components for PCR plus primer-bound beads and template DNA are stirred together with an oil/detergent mix to create microemulsions. The aqueous compartments (white circles in the gray oil layer) contain an average of less than one template molecule and less than one bead. Red and green templates represent two template molecules, the sequences of which differ by one or many nucleotides. Step 3: The microemulsions are temperature-cycled as in a conventional PCR. If a DNA template and a bead are present together in a single aqueous compartment, the bead-bound oligonucleotides act as primers for amplification. The straight red and green lines connected to the beads represent extension products from the two different kinds of templates. Step 4: The emulsions are broken, and the beads are purified with a magnet. Step 5: After denaturation, the beads are incubated with oligonucleotides that can distinguish between the sequences of the different kinds of templates. Fluorescently labeled antibodies then are used to label the bound hybridization probes, which renders the beads containing PCR product as red or green after appropriate laser excitation. Step 6: Flow cytometry is used to count the red and green beads.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations

    doi: 10.1073/pnas.1133470100

    Figure Lengend Snippet: Schematic of BEAMing. Step 1: Magnetic beads covalently coated with streptavidin are bound to biotinylated oligonucleotides (oligos). Step 2: An aqueous mix containing all the necessary components for PCR plus primer-bound beads and template DNA are stirred together with an oil/detergent mix to create microemulsions. The aqueous compartments (white circles in the gray oil layer) contain an average of less than one template molecule and less than one bead. Red and green templates represent two template molecules, the sequences of which differ by one or many nucleotides. Step 3: The microemulsions are temperature-cycled as in a conventional PCR. If a DNA template and a bead are present together in a single aqueous compartment, the bead-bound oligonucleotides act as primers for amplification. The straight red and green lines connected to the beads represent extension products from the two different kinds of templates. Step 4: The emulsions are broken, and the beads are purified with a magnet. Step 5: After denaturation, the beads are incubated with oligonucleotides that can distinguish between the sequences of the different kinds of templates. Fluorescently labeled antibodies then are used to label the bound hybridization probes, which renders the beads containing PCR product as red or green after appropriate laser excitation. Step 6: Flow cytometry is used to count the red and green beads.

    Article Snippet: They then were incubated for 10 min in a total volume of 100 μl of B-PCR buffer containing 3 μg of an Alexa 488-conjugated goat anti-chicken antibody (no. A-11039, Molecular Probes) and 3 μg of R-phycoerythrin-labeled streptavidin (no. S-866, Molecular Probes).

    Techniques: Magnetic Beads, Polymerase Chain Reaction, Activated Clotting Time Assay, Amplification, Purification, Incubation, Labeling, Hybridization, Flow Cytometry, Cytometry

    Affinity maturation of hu3F8 scFv by random mutagenesis. A , FACS for yeast display selection. The yeast library was labeled with mouse anti-c-Myc antibody followed by goat anti-mouse dye as well as biotinylated GD2 followed by streptavidin dye. During

    Journal: The Journal of Biological Chemistry

    Article Title: Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8 *

    doi: 10.1074/jbc.M115.650903

    Figure Lengend Snippet: Affinity maturation of hu3F8 scFv by random mutagenesis. A , FACS for yeast display selection. The yeast library was labeled with mouse anti-c-Myc antibody followed by goat anti-mouse dye as well as biotinylated GD2 followed by streptavidin dye. During

    Article Snippet: 1:100 dilutions of both R-phycoerythrin-conjugated streptavidin (Invitrogen) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Invitrogen) were added, incubated at 4 °C for 30 min, and washed three times with PBSA buffer for FACS selection.

    Techniques: Mutagenesis, FACS, Selection, Labeling

    Specific binding of WM-Myr47b peptide to cell surface tsNTCP. (A) 293T cells were transiently transfected with plasmids encoding tsNTCP-GFP or hSDC2-GFP as a control. Transfected cells were cultured in PMM for 36 to 48 h, blocked with 3% bovine serum albumin–phosphate-buffered saline for 1 h, and then incubated with the indicated peptides at 400 nM at 37°C for 2 h. Subsequently, the cells were fixed with 4% paraformaldehyde, stained with 0.6 μg/ml PE-streptavidin, and visualized with a Zeiss LSM 510 Meta confocal microscope. (B) 293T cells transfected with tsNTCP-GFP were blocked with 3% bovine serum albumin–phosphate-buffered saline in the absence (top) or presence (bottom) of 800 nM nonbiotinylated HBV pre-S1 peptide, H-Myr47, followed by washing and incubation with the biotinylated WMHBV pre-S1 peptide, WM-Myr47b, at 400 nM at 37°C for 2 h. Cells were then stained and visualized as described for panel A. DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Journal of Virology

    Article Title: Sodium Taurocholate Cotransporting Polypeptide Mediates Woolly Monkey Hepatitis B Virus Infection of Tupaia Hepatocytes

    doi: 10.1128/JVI.03533-12

    Figure Lengend Snippet: Specific binding of WM-Myr47b peptide to cell surface tsNTCP. (A) 293T cells were transiently transfected with plasmids encoding tsNTCP-GFP or hSDC2-GFP as a control. Transfected cells were cultured in PMM for 36 to 48 h, blocked with 3% bovine serum albumin–phosphate-buffered saline for 1 h, and then incubated with the indicated peptides at 400 nM at 37°C for 2 h. Subsequently, the cells were fixed with 4% paraformaldehyde, stained with 0.6 μg/ml PE-streptavidin, and visualized with a Zeiss LSM 510 Meta confocal microscope. (B) 293T cells transfected with tsNTCP-GFP were blocked with 3% bovine serum albumin–phosphate-buffered saline in the absence (top) or presence (bottom) of 800 nM nonbiotinylated HBV pre-S1 peptide, H-Myr47, followed by washing and incubation with the biotinylated WMHBV pre-S1 peptide, WM-Myr47b, at 400 nM at 37°C for 2 h. Cells were then stained and visualized as described for panel A. DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: The cells were then stained with phycoerythrin (PE)-conjugated streptavidin (eBioscience, San Diego, CA) to detect the biotin tag of the peptides.

    Techniques: Binding Assay, Transfection, Cell Culture, Incubation, Staining, Microscopy

    A representative FACS analysis of ocular infiltrating cells before v after PC. A flow cytometric analysis was carried out on each ocular infiltration inflammatory cells. Left panel: The discrimination of ocular infiltrating cells by forward and side scattered gate. Only viable cells were analysed in the live gate. Typical neutrophil and macrophage gate and NK cell and lymphocyte gate were indicated. Right panel: Neutrophil and macrophage: The rectangular gates indicated neutrophils and macrophages. Neutrophils were stained with FITC conjugated anti-Gr-1 mAb and Cy-chrome conjugated anti-CD45 mAb. Macrophages were stained with biotin conjugated anti-F4/80 monoclonal antibody (mAb) counterstained by Streptavidin/R-PE and Cy-chrome conjugated anti-CD45 mAb. Lymphocyte: The quadrant gate indicated T cells and B cells. Cells were stained with Cy-chrome conjugated anti-mouse TCR β chain mAb (T cells) and FITC conjugated anti-mouse CD19 mAb (B cells). NK cell: NK cells were indicated by the histogram. Cells were stained with and biotin conjugated anti-mouse NK1. 1 mAb counter stained by Streptavidin/R-PE.

    Journal: The British Journal of Ophthalmology

    Article Title: The relative contributions of each subset of ocular infiltrated cells in experimental choroidal neovascularisation

    doi: 10.1136/bjo.2003.036392

    Figure Lengend Snippet: A representative FACS analysis of ocular infiltrating cells before v after PC. A flow cytometric analysis was carried out on each ocular infiltration inflammatory cells. Left panel: The discrimination of ocular infiltrating cells by forward and side scattered gate. Only viable cells were analysed in the live gate. Typical neutrophil and macrophage gate and NK cell and lymphocyte gate were indicated. Right panel: Neutrophil and macrophage: The rectangular gates indicated neutrophils and macrophages. Neutrophils were stained with FITC conjugated anti-Gr-1 mAb and Cy-chrome conjugated anti-CD45 mAb. Macrophages were stained with biotin conjugated anti-F4/80 monoclonal antibody (mAb) counterstained by Streptavidin/R-PE and Cy-chrome conjugated anti-CD45 mAb. Lymphocyte: The quadrant gate indicated T cells and B cells. Cells were stained with Cy-chrome conjugated anti-mouse TCR β chain mAb (T cells) and FITC conjugated anti-mouse CD19 mAb (B cells). NK cell: NK cells were indicated by the histogram. Cells were stained with and biotin conjugated anti-mouse NK1. 1 mAb counter stained by Streptavidin/R-PE.

    Article Snippet: Streptavidin/R-PE was purchased from Molecular Probes (Oregon, USA).

    Techniques: FACS, Flow Cytometry, Staining

    The absence of NF-κB1 accelerates apoptosis of quiescent B cells. ( A ) B cells turnover more rapidly in nfkb1 −/− mice. The turnover of virgin and mature splenic B cells was determined by BrdU incorporation. Splenic B cells isolated from normal, c-rel −/− , and nfkb1 −/− mice fed with BrdU for 2, 4, or 7 d were subjected to three-color immunofluorescence staining, after which flow cytometric analysis was used to identify virgin (sIgM + sIgD − ) and mature (sIgM + sIgD + ) B cells and to determine the fraction of these cells that had incorporated BrdU. Examples from the analysis of mice fed BrdU for 4 d are shown. The top row shows two-color FACS ® dot plots ( x-axis , staining with R-PE–labeled anti-IgM antibodies; y-axis , staining with biotinylated anti-IgD antibodies plus Tricolor-streptavidin), with the boxed regions used for electronic gating of mature B cells. The middle rows present histograms of the anti-BrdU–labeling intensity (FITC) of these mature B cells. The percentages of BrdU + cells are indicated as are the boundaries used for distinguishing them from BrdU − cells. The bottom panels summarize the kinetics of BrdU labeling for virgin and mature splenic B cells over a 4- and 7-d time course, respectively. The values are arithmetic means plus SD from the analysis of three normal, c-rel −/− , and nfkb1 −/− mice at each time point. ( B ) Cell death in culture. Resting splenic B cells from normal ( triangles ), c-rel −/− ( circles ), and nfkb1 −/− ( squares ) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and flow cytometric analysis of fixed cells stained with PI. At the start of the experiment, > 99% of cells of all genotypes were viable. The data represents the mean ±SD of five experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: B Lymphocytes Differentially Use the Rel and Nuclear Factor ?B1 (NF-?B1) Transcription Factors to Regulate Cell Cycle Progression and Apoptosis in Quiescent and Mitogen-activated Cells

    doi:

    Figure Lengend Snippet: The absence of NF-κB1 accelerates apoptosis of quiescent B cells. ( A ) B cells turnover more rapidly in nfkb1 −/− mice. The turnover of virgin and mature splenic B cells was determined by BrdU incorporation. Splenic B cells isolated from normal, c-rel −/− , and nfkb1 −/− mice fed with BrdU for 2, 4, or 7 d were subjected to three-color immunofluorescence staining, after which flow cytometric analysis was used to identify virgin (sIgM + sIgD − ) and mature (sIgM + sIgD + ) B cells and to determine the fraction of these cells that had incorporated BrdU. Examples from the analysis of mice fed BrdU for 4 d are shown. The top row shows two-color FACS ® dot plots ( x-axis , staining with R-PE–labeled anti-IgM antibodies; y-axis , staining with biotinylated anti-IgD antibodies plus Tricolor-streptavidin), with the boxed regions used for electronic gating of mature B cells. The middle rows present histograms of the anti-BrdU–labeling intensity (FITC) of these mature B cells. The percentages of BrdU + cells are indicated as are the boundaries used for distinguishing them from BrdU − cells. The bottom panels summarize the kinetics of BrdU labeling for virgin and mature splenic B cells over a 4- and 7-d time course, respectively. The values are arithmetic means plus SD from the analysis of three normal, c-rel −/− , and nfkb1 −/− mice at each time point. ( B ) Cell death in culture. Resting splenic B cells from normal ( triangles ), c-rel −/− ( circles ), and nfkb1 −/− ( squares ) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and flow cytometric analysis of fixed cells stained with PI. At the start of the experiment, > 99% of cells of all genotypes were viable. The data represents the mean ±SD of five experiments.

    Article Snippet: Binding of biotinylated antibodies was revealed by staining with R-phycoerythrin-streptavidin (CALTAG Labs.) as the secondary reagent.

    Techniques: Mouse Assay, BrdU Incorporation Assay, Isolation, Immunofluorescence, Staining, Flow Cytometry, FACS, Labeling, Cell Culture

    Effects of hybridization temperature (A), time (B), streptavidin-R-phycoerythrin (SA-PE) concentrations (C), and wash conditions (D) on hybridization of C. hominis (hatched bars) and C. parvum (white bars) capture probes to ML-2 amplicons. The bars represent

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid Microsphere Assay for Identification of Cryptosporidium hominis and Cryptosporidium parvum in Stool and Environmental Samples ▿

    doi: 10.1128/JCM.00138-07

    Figure Lengend Snippet: Effects of hybridization temperature (A), time (B), streptavidin-R-phycoerythrin (SA-PE) concentrations (C), and wash conditions (D) on hybridization of C. hominis (hatched bars) and C. parvum (white bars) capture probes to ML-2 amplicons. The bars represent

    Article Snippet: After incubation, the plate was centrifuged at 3,580 × g for 5 min, 25 μl of supernatant was carefully removed, and 75 μl of a 1:40 dilution of streptavidin-R-phycoerythrin (SA-PE) (Molecular Probes, Eugene, OR) was added.

    Techniques: Hybridization

    Validation of E. coli whole-cell biosensors. ( a ) Biosensor circuit design and localisation in the outer membrane of the cell. ( b ) Induction of biosensor expression. Representative cell pellets (OD 600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. ( c ) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. ( d ) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. ( e ) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P

    Journal: Scientific Reports

    Article Title: A protease-based biosensor for the detection of schistosome cercariae

    doi: 10.1038/srep24725

    Figure Lengend Snippet: Validation of E. coli whole-cell biosensors. ( a ) Biosensor circuit design and localisation in the outer membrane of the cell. ( b ) Induction of biosensor expression. Representative cell pellets (OD 600 4.0) of either induced 100 mM xylose (+) or uninduced (−) biosensor-expressing cells were labelled with 1.25 μg streptavidin-R-phycoerythrin (SAPE)-conjugated antibody. Cell labelling (Red) indicates appropriate expression and localisation of the whole-cell biosensor. ( c ) Flow cytometry analysis of whole-cell biosensors. E. coli expressing either TEV (mCPX-TEV), elastase (mCPX-ELA) or control (mCPX-CON) biosensors were treated with the indicated proteases: AcTEV protease (TEV) or control proteases - PreScission protease (PRE) or Enterokinase (ENT). Treated cells were labelled with SAPE-conjugated antibody and analysed by flow cytometry. Labelled, non-protease treated cells and E. coli transformed with an empty vector plasmid (EV) served as experimental controls. ( d ) Summary of flow cytometry data. E. coli expressing either mCPX-TEV, mCPX-ELA or mCPX-CON biosensors were treated with the indicated proteases. The fluorescence (Geometric mean FL5) of protease treated cells were normalised against labelled, non-protease treated cells (No Prot). These data were analysed using FlowJo (vX 10.0.7r2) software and are representative of three independent biological repeats. ( e ) Sensitivity of mCPX-TEV biosensor. E. coli expressing mCPX-TEV biosensor were treated with 0–10 Units of AcTEV and analysed via flow cytometry. These data are normalised against untreated (0 U) labelled cells and represent the mean geometric mean ± the standard deviation of three independent experiments. Student t-test ****P

    Article Snippet: E. coli cultures were then labelled by the addition of 1.25 μg SAPE (SNN1007, Life technologies, UK), whilst B. subtilis cultures were labelled by the addition of 2.5 μg His-PE (ab72467, Abcam, UK).

    Techniques: Expressing, Flow Cytometry, Cytometry, Transformation Assay, Plasmid Preparation, Fluorescence, Software, Standard Deviation

    Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5  Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.

    Journal: PLoS ONE

    Article Title: T0001, a variant of TNFR2-Fc fusion protein, exhibits improved Fc effector functions through increased binding to membrane-bound TNFα

    doi: 10.1371/journal.pone.0177891

    Figure Lengend Snippet: Competitive binding of TNFα antagonists to FcγRIIIa on transfected cells. 2×10 5 Jurkat cells transfected with FcγRIIIa(158V) were incubated with TNFα antagonists competed with 6.7 nM biotin-IgG1 alone or in the presence of 1:1 molar excess sTNFα for 0.5 h at 4°C, followed by staining with Streptavidin R-PE Conjugate as a secondary antibody for 0.5 h at 4°C. The binding of TNFα antagonists to FcγRIIIa expressed as the percentage competition as described in “Materials and methods”.

    Article Snippet: The cells were washed with PBS to remove unbound agents and stained with Streptavidin R-PE Conjugate (Life Technologies) as a secondary antibody for 0.5 h at 4°C.

    Techniques: Binding Assay, Transfection, Incubation, Staining

    Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’) 2 generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate as the target cell and peripheral blood mononuclear cells PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cell in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and streptavidin-PE conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1

    Journal: Breast Cancer Research : BCR

    Article Title: Proteolytic single hinge cleavage of pertuzumab impairs its Fc effector function and antitumor activity in vitro and in vivo

    doi: 10.1186/s13058-018-0972-4

    Figure Lengend Snippet: Anti-hinge antibodies rescued antibody dependent cellular cytotoxicity (ADCC) activity for single hinge cleaved pertuzumab (scIgG-P). a-b Purified human anti-protease-induced, anti-hinge autoantibodies (AHA) using peptide analogues representing hinge-immunoglobulin G-degrading enzyme S (IdeS) cleavage sites, 1981 or F(ab’) 2 generated by digesting immunoglobulin G (IgG-P) with IdeS as the absorbent, restored ADCC activity for scIgG-P. SKOV-3 cell (5000 cells/well) was seeded on the E-plate as the target cell and peripheral blood mononuclear cells PBMCs (25,000 cells/well) isolated from a single donor were used as the immune effector cell in complete cell culture medium containing scIgG-P (30 nM). The percentage of cell lysis was defined as: (cell index of control group – cell index of treatment group)/cell index of control group) × 100. c Flow cytometry showing binding results for AH-mAb with IgG-P or scIgG-P on surfaces of high human epidermal growth factor receptor 2-expressing cancer cells. Biotinylated 2095–2 and streptavidin-PE conjugate were used for cell staining. d-e 2095–2 ADCC rescuing effect for scIgG-P at varying concentrations. A fixed concentration of 30 nM for IgG-P with threefold dilutions from 30 nM for 2095–2 were used in the ADCC assay. SKOV-3 cells (5000 cells/well) and SKBR3 cell (7000 cells/well) were used as the target cells and PBMCs isolated from a single donor were used as the immune effector cells at an effector (E)-target (T) ratio of 25:1

    Article Snippet: For the determination of the anti-hinge antibody binding to scIgG-P and scIgG-T on cancer cell surfaces, AHA (mAb 2095–2) was biotinylated and the binding of the AHA was detected using R-PE conjugated streptavidin (1:200) (Jackson Immune Research laboratories).

    Techniques: Activity Assay, Purification, Generated, Isolation, Cell Culture, Lysis, Flow Cytometry, Cytometry, Binding Assay, Expressing, Staining, Concentration Assay, ADCC Assay

    Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Acquisition of anti-CD19 chimeric antigen receptors (CARs) by NK cells from donor cells via trogocytosis. A. Flow cytometry dot plots illustrating the uptake of anti-CD19 CARs by NK cells via trogocytosis. NK cells co-cultured with K562 cells (control) or K562-antiCD19BBζ cells were stained with an anti-CD56-FITC antibody and a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by PE-conjugated streptavidin. B. Uptake of anti-CD19 CARs by NK cells expanded by co-culturing with irradiated (IR) or freeze/thaw-treated (F) K562-mb15-41BBL cells. The data are presented as the mean ± SD of values obtained using three unrelated NK donors. C. Kinetics of anti-CD19 CAR uptake by NK cells from K562-antiCD19BBζ cells (black bars) and control K562 cells (white bars). The uptake of anti-CD19 CARs by NK cells was analyzed after co-culturing with feeder cells for the indicated time and was compared with that of NK cells co-cultured with control K562 cells. The data are presented as the mean ± SD of values obtained using 3 unrelated NK cell donors. *: significant increase compared with control cells (p

    Article Snippet: Biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072) and PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) were used for labeling the cells.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Staining, Irradiation

    Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Journal: PLoS ONE

    Article Title: Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

    doi: 10.1371/journal.pone.0109352

    Figure Lengend Snippet: Immunofluorescence analysis for trogocytosis. A. NK cells stained with anti-CD56-FITC antibody. B. K562-antiCD19BBζ cells were stained with a biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment specific antibody, followed by Alexa Fluor 568-conjugated streptavidin. The nucleus was stained with DAPI (blue). C. NK cells co-cultured with K562-antiCD19BBζ cells were stained for CD56 and anti-CD19 CARs, as previously described. D. Acquisition of anti-CD19-BB-ζ by NK cells via trogocytosis was observed.

    Article Snippet: Biotin-SP-conjugated AffiniPure goat anti-mouse IgG,F(ab′)2 fragment-specific antibody (Jackson ImmunoResearch 115-065-072) and PE-conjugated streptavidin (Jackson ImmunoResearch 016-110-084) were used for labeling the cells.

    Techniques: Immunofluorescence, Staining, Cell Culture

    Hybridization of the InflA probe, with and without an increasing number of dInosines, with targets containing increasing numbers of mismatching nucleotides. The sequence of the InflA probe was designed from a conserved region of the matrix 2 gene in segment 7 of subtype H2N3 of the Influenza A virus ( Figure 1 , and Table 1 sequence of H3N2). The number of dInosines included in the probes is indicated in the names Ino3–Ino21. Similarly, the wobbN_21 probe contains 21 N wobbles in the same position as the dInosines in the Ino21 probe. The complementary target of the InflA probe is named the InflA target. These targets have 0–21 pm, as indicated by the names; the 21-gm target has seven groups of three mismatches ( Figure 2 D). All target molecules were biotinylated at their 5′ end. Each sample contained 0.2 nM of one of the synthetic biotinylated single-stranded targets and a mixture containing 2500 microspheres of each of the unique coupled probes tested. In ( A ) and ( B ), the mixture contained the InflA probe and the Ino3, 5, 7, 9 and 21 probes; in ( C ), the mixture contained the InflA probe, the Ino21 probe and the wobbN_21 probe. Hybridization was performed at 45°C (A and C) and 55°C (B and C) in a buffer containing 3 M TMAC for 30 min, followed by 15-min incubation with Streptavidin-R-phycoerythrin. A total of 100 microspheres per type of probe were analyzed in the Luminex 200; the extent of hybridization is indicated as median fluorescent intensity (MFI) on the y-axis. Each experiment contained triplet samples and was repeated two or three times. The sequences of probes and targets are provided in Supplementary Table 1A and B . ( D ) The figure shows the distribution of dInosines (orange/light blue/green boxes) and N wobbles (light grey boxes) in the probes (grey bar), and the mismatching bases (magenta boxes) in the target molecules (black bar), used in Figure 2 A–C.

    Journal: Nucleic Acids Research

    Article Title: Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

    doi: 10.1093/nar/gkq777

    Figure Lengend Snippet: Hybridization of the InflA probe, with and without an increasing number of dInosines, with targets containing increasing numbers of mismatching nucleotides. The sequence of the InflA probe was designed from a conserved region of the matrix 2 gene in segment 7 of subtype H2N3 of the Influenza A virus ( Figure 1 , and Table 1 sequence of H3N2). The number of dInosines included in the probes is indicated in the names Ino3–Ino21. Similarly, the wobbN_21 probe contains 21 N wobbles in the same position as the dInosines in the Ino21 probe. The complementary target of the InflA probe is named the InflA target. These targets have 0–21 pm, as indicated by the names; the 21-gm target has seven groups of three mismatches ( Figure 2 D). All target molecules were biotinylated at their 5′ end. Each sample contained 0.2 nM of one of the synthetic biotinylated single-stranded targets and a mixture containing 2500 microspheres of each of the unique coupled probes tested. In ( A ) and ( B ), the mixture contained the InflA probe and the Ino3, 5, 7, 9 and 21 probes; in ( C ), the mixture contained the InflA probe, the Ino21 probe and the wobbN_21 probe. Hybridization was performed at 45°C (A and C) and 55°C (B and C) in a buffer containing 3 M TMAC for 30 min, followed by 15-min incubation with Streptavidin-R-phycoerythrin. A total of 100 microspheres per type of probe were analyzed in the Luminex 200; the extent of hybridization is indicated as median fluorescent intensity (MFI) on the y-axis. Each experiment contained triplet samples and was repeated two or three times. The sequences of probes and targets are provided in Supplementary Table 1A and B . ( D ) The figure shows the distribution of dInosines (orange/light blue/green boxes) and N wobbles (light grey boxes) in the probes (grey bar), and the mismatching bases (magenta boxes) in the target molecules (black bar), used in Figure 2 A–C.

    Article Snippet: An amount of 2 µl (0.05 mg/ml) of streptavidin-R-phycoerythrin (QIAGEN, Hilden, Germany) was added to the mixture, which was further incubated at 45 or 55°C for 15 min before analysis for internal microsphere and R-phycoerythrin reporter fluorescence on the Luminex®200™ system (Luminex corporation, Austin, TX).

    Techniques: Hybridization, Sequencing, Incubation, Luminex

    Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B 12 . BtuCD WT denotes the wild-type, BtuCD cys denotes the cysteine mutant and BtuCD EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B 12 to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a biotin-streptavidin link and imaged using TIRF microscopy. d A complex of BtuCD cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg 2+ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c , but with the ATPase impaired mutant BtuCD EQ . No events were observed. f Similar experiment as described in c , but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d – f around 1000 single-molecule fluorescence traces were analysed

    Journal: Nature Communications

    Article Title: Single-molecule visualization of conformational changes and substrate transport in the vitamin B12 ABC importer BtuCD-F

    doi: 10.1038/s41467-017-01815-7

    Figure Lengend Snippet: Experimental setup, ATPase rate and fluorescence quenching by ATP and BtuF. a Structure of BtuF (pink) bound to BtuCD (BtuC homodimer: green, BtuD homodimer: blue) in the absence of nucleotide and substrate (Protein Data Bank (PDB) ID 2QI9). Cysteine mutations for labelling in BtuF (D141C) and BtuC (Q111C) are marked with a yellow dot. The distance between the labelling positions is ~37 Å. b ATPase rate of various BtuCD-F mutants with or without fluorescent label reconstituted in liposomes and loaded with (pink bar) or without (grey bar) vitamin B 12 . BtuCD WT denotes the wild-type, BtuCD cys denotes the cysteine mutant and BtuCD EQ denotes the cysteine mutant that is ATPase-impaired. For all combinations, the cysteine mutant of BtuF is used. Measured rates are not corrected for orientation of the transporter. When BtuF is present at the concentrations used, the full complex is formed. Values displayed are the mean and standard deviation of three experiments. c Experimental design (fluorescent labels are omitted). BtuCD was reconstituted in liposomes of 100-nm diameter in ratios such that on average one transporter was found per liposome. By introducing BtuF and vitamin B 12 to the lumen of the vesicle and ATP on the outside or vice versa, only one particular orientation of the transporter was probed. Proteoliposomes are tethered to a glass surface via a biotin-streptavidin link and imaged using TIRF microscopy. d A complex of BtuCD cys labelled with Alexa Fluor 555 and unlabelled BtuF showed decrease in fluorescence intensity upon addition of 2 mM ATP and 10 mM Mg 2+ on the outside (middle panel). The distribution of event times of the first drop of intensity is plotted in a histogram (right panel) for the positive (pos, with ATP) and negative (neg, without ATP) experiment. For a description of the data analysis, see methods. e Similar experiment as described in c , but with the ATPase impaired mutant BtuCD EQ . No events were observed. f Similar experiment as described in c , but the vesicle lumen was left empty. Upon introduction of BtuF to the outside of the liposomes an increase in fluorescence intensity was observed. For each condition in d – f around 1000 single-molecule fluorescence traces were analysed

    Article Snippet: Following this step, 0.1 mg ml−1 streptavidin (streptavidin from Streptomyces avidinii , Sigma-Aldrich) in buffer C was incubated for 5 min.

    Techniques: Fluorescence, Mutagenesis, Standard Deviation, Microscopy

    LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by streptavidin-PE.

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: LPS down-regulates MTS510 binding to cell surface TLR4-MD-2. Ba/F3 cells expressing TLR4-MD-2 (left) or CD14 + TLR4-MD-2 (middle), or a macrophage line RAW264 (right) were stimulated with medium alone, 1 μg/ml lipid A, 10 μg/ml peptidoglycan (PGN), or 100 μM CpG DNA as indicated. After washing, cells were stained with biotinylated MTS510 followed by streptavidin-PE.

    Article Snippet: Cells were washed in staining buffer, and incubated with R-PE–conjugated goat anti–rat IgG Ab (Southern Biotechnology Associates Inc.), FITC-conjugated goat anti–mouse IgG (BD Biosciences), or R-PE–conjugated streptavidin (BD Biosciences) for 15 min.

    Techniques: Binding Assay, Expressing, Staining

    E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: E5531 acts on LPS interaction with TLR4-MD-2 at a concentration that does not affect LPS binding to mCD14. (a) Ba/F3 cells expressing TLR4-MD-2 and CD14 were pretreated with or without E5531 (indicated concentration) at 37°C for 30 min. Cells were stimulated with medium alone or 3 μg/ml LPS at 37°C for 30 min. After washing, cells were stained with biotinylated MTS510 mAb followed by streptavidin-PE (left and middle columns), or with anti-LPS followed by goat anti–mouse IgG-FITC (right column). Open histograms depict staining with the secondary reagent alone. (b) 3 μg/ml LPS with indicated concentrations of E5531 was added to the supernatant from Ba/F3 cells expressing CD14. sCD14 in the supernatant was precipitated with anti-CD14 mAb, followed by immunoprobing with anti-LPS (top) or anti-CD14 (bottom). (c) After treatment with E5531 and LPS as in panel a, cells were subjected to cell surface biotinylation, washing, detergent lysis, immunoprecipitation with Sa15-21, SDS-PAGE (polyacrylamide gel:18.0% for LPS and 7.5% for TLR4; under nonreducing conditions), and electroblotting. Precipitated LPS (top) and cell surface TLR4 (bottom) were probed with anti-LPS mAb or alkaline phosphatase–conjugated streptavidin, respectively. (d) After treatment with LPS and E5531 as in panel c, cells were subjected to detergent lysis, SDS-PAGE, electroblotting, and immunoprobing IkBα (top) or actin (bottom).

    Article Snippet: Cells were washed in staining buffer, and incubated with R-PE–conjugated goat anti–rat IgG Ab (Southern Biotechnology Associates Inc.), FITC-conjugated goat anti–mouse IgG (BD Biosciences), or R-PE–conjugated streptavidin (BD Biosciences) for 15 min.

    Techniques: Concentration Assay, Binding Assay, Expressing, Staining, Lysis, Immunoprecipitation, SDS Page

    A novel mAb to TLR4-MD-2 reveals the LPS-triggered change of cell surface TLR4-MD-2. (a) Immunoprecipitation with anti-flag (top) or Sa15-21 (bottom) was conducted with Ba/F3 transfectants expressing the indicated molecules (Materials and Methods). The precipitates were probed with rabbit anti–mouse TLR4 sera followed by goat anti–rabbit alkaline phosphatase. Only immature, smaller TLR4 is detected in cells expressing TLR4 alone (top, TLR4f and CD14/TLR4f), because TLR4 without MD-2 cannot reach the cell surface. (b) Ba/F3 transfectants expressing CD14 and TLR4-MD-2 were stimulated with medium alone, 1 μg/ml lipid A, or 1 μg/ml LPS at 37°C for 30 min. Cells were stained with biotinylated MTS510 mAb or Sa15-21 as indicated, followed by streptavidin-PE. Open histograms depict staining with streptavidin-PE alone. (c) Ba/F3 cells expressing TLR4-MD-2 and CD14 (top) or RAW264 (bottom) were stimulated with medium, 2 μg/ml LPS, or 2 μg/ml lipid A antagonist E5531 as indicated at 37°C for 30 min. After washing, cells were subjected to cell surface biotinylation, detergent lysis, immunoprecipitation with MTS510 mAb (right three lanes) or Sa15-21 mAb (left three lanes), SDS-PAGE (7.5% polyacrylamide under nonreducing conditions), and electroblotting. Precipitated cell surface TLR4 was probed with streptavidin–alkaline phosphatase conjugate.

    Journal: The Journal of Experimental Medicine

    Article Title: Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2

    doi: 10.1084/jem.20031076

    Figure Lengend Snippet: A novel mAb to TLR4-MD-2 reveals the LPS-triggered change of cell surface TLR4-MD-2. (a) Immunoprecipitation with anti-flag (top) or Sa15-21 (bottom) was conducted with Ba/F3 transfectants expressing the indicated molecules (Materials and Methods). The precipitates were probed with rabbit anti–mouse TLR4 sera followed by goat anti–rabbit alkaline phosphatase. Only immature, smaller TLR4 is detected in cells expressing TLR4 alone (top, TLR4f and CD14/TLR4f), because TLR4 without MD-2 cannot reach the cell surface. (b) Ba/F3 transfectants expressing CD14 and TLR4-MD-2 were stimulated with medium alone, 1 μg/ml lipid A, or 1 μg/ml LPS at 37°C for 30 min. Cells were stained with biotinylated MTS510 mAb or Sa15-21 as indicated, followed by streptavidin-PE. Open histograms depict staining with streptavidin-PE alone. (c) Ba/F3 cells expressing TLR4-MD-2 and CD14 (top) or RAW264 (bottom) were stimulated with medium, 2 μg/ml LPS, or 2 μg/ml lipid A antagonist E5531 as indicated at 37°C for 30 min. After washing, cells were subjected to cell surface biotinylation, detergent lysis, immunoprecipitation with MTS510 mAb (right three lanes) or Sa15-21 mAb (left three lanes), SDS-PAGE (7.5% polyacrylamide under nonreducing conditions), and electroblotting. Precipitated cell surface TLR4 was probed with streptavidin–alkaline phosphatase conjugate.

    Article Snippet: Cells were washed in staining buffer, and incubated with R-PE–conjugated goat anti–rat IgG Ab (Southern Biotechnology Associates Inc.), FITC-conjugated goat anti–mouse IgG (BD Biosciences), or R-PE–conjugated streptavidin (BD Biosciences) for 15 min.

    Techniques: Immunoprecipitation, Expressing, Staining, Lysis, SDS Page