alkaline phosphatase-conjugated streptavidin Search Results


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  • 99
    Vector Laboratories alkaline phosphatase conjugated streptavidin
    Effect of the T DnaK⋅J-GrpE set and T ClpB on aggregation of LDH. LDH, biotinylated for detection, was incubated in the presence of indicated components at 73°C for 30 min. The solutions were centrifuged, and supernatant (sup) and precipitate (ppt) were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Electrophoresed proteins were blotted to the membrane, and biotinylated LDH was detected by alkaline phosphatase-conjugated <t>streptavidin.</t> KJ, E, and B represent T DnaK⋅J complex, T GrpE and T ClpB, respectively.
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher alkaline phosphatase conjugated streptavidin
    Surface plasmon resonance analysis of biotin-tubulin binding to <t>strepavidin</t> and subsequent dimer dissociation as a result of dilution. (A) The plasmon resonance signal was increased by 954 RU during a 10-min flow of biotin-tubulin in Pi buffer with 12 mM Mg. The almost instantaneous 3000 RU signal change at the start and finish of the flow of the tubulin resulted from a difference in refractive index of the tubulin solution and the buffer. (B) Flow of tubulin- and nucleotide-free buffer resulted in a 445 RU signal decrease; the curve corresponds to a rate constant 14.72 × 10 −5 s −1 . A rate constant equal to 12.35 × 10 −5 s −1 was determined from a Guggenheim plot of the data.
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jackson Immuno alkaline phosphatase conjugated streptavidin
    Immunoprecipitation and immunoblotting with anti-L* Ab. Multiple 10-μm-thick deparaffinized brain tissue sections from a DA-infected SJL/J mouse (3 days p.i.) were used for protein extraction. The protein extracted from the brain sections of the uninfected SJL/J mouse was used as a negative control, and the protein extracted from BHK-21 cells infected with DA was used as a positive control. L* was immunoprecipitated from the extracted protein by affinity-purified L* Ab bound to the protein G and immobilized by cross-linker. Immnoprecipitated proteins were separated on an SDS-polyacrylamide gel. The bound Ig (anti-L* Ab) was detected with biotinylated secondary Abs and alkaline phosphatase-conjugated <t>streptavidin</t> using BCIP/NBT. Lanes: 1, SJL/J mouse infected with DA; 2, uninfected SJL/J mouse; 3, BHK-21 cells infected with DA. The arrow indicates the immunoprecipitated L*.
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore alkaline phosphatase conjugated streptavidin
    Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and <t>streptavidin</t> linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Agilent technologies alkaline phosphatase conjugated streptavidin
    Characterisation of ppIgG. (A) Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab) 2 antibody, followed by alkaline phosphatase-coupled <t>streptavidin</t> (see Materials and Methods ). (B) Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, E. coli O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. (C) ppIgG was tested for reactivity against Escherichia coli and Salmonella diarizonae by competitive ELISA as described ( Materials and Methods ). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 328 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mab Technologies streptavidin alkaline phosphatase conjugate
    Characterisation of ppIgG. (A) Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab) 2 antibody, followed by alkaline phosphatase-coupled <t>streptavidin</t> (see Materials and Methods ). (B) Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, E. coli O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. (C) ppIgG was tested for reactivity against Escherichia coli and Salmonella diarizonae by competitive ELISA as described ( Materials and Methods ). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p
    Streptavidin Alkaline Phosphatase Conjugate, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 92/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher streptavidin alkaline phosphatase conjugate
    Biotinylated DNA tether binds <t>streptavidin.</t> (A) Schematic of the double-stranded DNA tether prepared from half lambda DNA that contains functional groups as indicated on both ends of the molecule. (B) A representative force-extension curve for a single DNA tether formed between an anti-digoxigenin coated polystyrene particle and a biotin-coated polystyrene particle. The length of the overstretch transition in this figure measures 5.4 |im, consistent with that of a half-lambda DNA.
    Streptavidin Alkaline Phosphatase Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mab Technologies streptavidin conjugated alkaline phosphatase
    Biotinylated DNA tether binds <t>streptavidin.</t> (A) Schematic of the double-stranded DNA tether prepared from half lambda DNA that contains functional groups as indicated on both ends of the molecule. (B) A representative force-extension curve for a single DNA tether formed between an anti-digoxigenin coated polystyrene particle and a biotin-coated polystyrene particle. The length of the overstretch transition in this figure measures 5.4 |im, consistent with that of a half-lambda DNA.
    Streptavidin Conjugated Alkaline Phosphatase, supplied by Mab Technologies, used in various techniques. Bioz Stars score: 92/100, based on 237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare alkaline phosphatase conjugated streptavidin
    TX-100 solubility of membrane proteins in HA/NA and HAt−/NAt− virus-infected MDCK cells. Influenza virus-infected MDCK cells were pulse-labeled with [ 35 S]-Promix, cell surfaces were biotinylated, cells were extracted with 1% TX-100, and soluble (S) and insoluble (I) fractions were separated by centrifugation. Proteins were immunoprecipitated, biotinylated proteins were recovered with <t>streptavidin-Sepharose</t> beads, and polypeptides were analyzed by SDS-PAGE. (A) Biotinylated HA and NA; (B) biotinylated HA and M 2 ; (C) total HA and M 1 immunoprecipitated using anti-A/Udorn/72 influenza virus serum; (D) quantitation of TX-100-insoluble HA, NA, M 1 , and M 2 proteins (data averaged from two independent experiments).
    Alkaline Phosphatase Conjugated Streptavidin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson alkaline phosphatase conjugated streptavidin
    CD72 CTLD specifically binds to Sm/RNP. (A–E) Conventional ELISA. Biotinylated CD72 a and CD72 c CTLD proteins at the indicated concentrations were incubated with ELISA plates coated with the indicated molecules. CD72 CTLD proteins bound to the ELISA plates were detected using alkaline phosphatase–conjugated <t>streptavidin</t> and phosphatase substrate. Data are representative of five independent experiments. (F and G) Competitive ELISA. (F) Binding of biotinylated CD72 c CTLD to Sm/RNP in the presence of various concentrations of unbiotinylated CD72 a and CD72 c CTLD proteins was measured. Representative data of five independent experiments are shown. (G) Mean ± SD of percent inhibition of binding in the presence of 100 µg/ml of the indicated competitors in triplicate is shown. **, P
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher streptavidin alkaline phosphatase
    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after <t>streptavidin-gold</t> labeling ( e ) or Ni-NTA-gold labeling ( f ).
    Streptavidin Alkaline Phosphatase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biogenex alkaline phosphatase conjugated streptavidin
    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after <t>streptavidin-gold</t> labeling ( e ) or Ni-NTA-gold labeling ( f ).
    Alkaline Phosphatase Conjugated Streptavidin, supplied by Biogenex, used in various techniques. Bioz Stars score: 92/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim streptavidin conjugated alkaline phosphatase
    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after <t>streptavidin-gold</t> labeling ( e ) or Ni-NTA-gold labeling ( f ).
    Streptavidin Conjugated Alkaline Phosphatase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega streptavidin conjugated alkaline phosphatase
    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after <t>streptavidin-gold</t> labeling ( e ) or Ni-NTA-gold labeling ( f ).
    Streptavidin Conjugated Alkaline Phosphatase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher streptavidin conjugated to alkaline phosphatase
    In vitro binding of PABP to coronavirus 3′ UTR RNAs. In vitro-translated luciferase or PABP was incubated with 1 μg of biotinylated BCV3′UTR RNAs containing poly(A) tails of 1, 5, 10, or 68 A residues (A) or MHV3′UTR RNAs containing poly(A) tails of 0, 5, 10, or > 50 A residues (B). Immobilized <t>streptavidin</t> was added to recover biotinylated RNA complexes, and samples were washed to remove any unbound RNA or protein. Samples were analyzed by SDS-PAGE (8% polyacrylamide). M (lanes 1 and 12) denotes marker and corresponds to the input amount of radiolabeled luciferase (lane 1) or PABP (lane 12) in each reaction. Lanes 2 and 7 represent the level of background protein binding in the absence of biotinylated RNA for luciferase (lane 2) and PABP (lane 7). (A) Lanes 3 to 6, luciferase recovered from interaction with BCV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with BCV3′UTR RNAs. (B) Lanes 3 to 6, luciferase recovered from interaction with MHV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with MHV3′UTR RNAs.
    Streptavidin Conjugated To Alkaline Phosphatase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effect of the T DnaK⋅J-GrpE set and T ClpB on aggregation of LDH. LDH, biotinylated for detection, was incubated in the presence of indicated components at 73°C for 30 min. The solutions were centrifuged, and supernatant (sup) and precipitate (ppt) were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Electrophoresed proteins were blotted to the membrane, and biotinylated LDH was detected by alkaline phosphatase-conjugated streptavidin. KJ, E, and B represent T DnaK⋅J complex, T GrpE and T ClpB, respectively.

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

    Article Title: Heat-inactivated proteins are rescued by the DnaK?J-GrpE set and ClpB chaperones

    doi:

    Figure Lengend Snippet: Effect of the T DnaK⋅J-GrpE set and T ClpB on aggregation of LDH. LDH, biotinylated for detection, was incubated in the presence of indicated components at 73°C for 30 min. The solutions were centrifuged, and supernatant (sup) and precipitate (ppt) were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Electrophoresed proteins were blotted to the membrane, and biotinylated LDH was detected by alkaline phosphatase-conjugated streptavidin. KJ, E, and B represent T DnaK⋅J complex, T GrpE and T ClpB, respectively.

    Article Snippet: Electrophoresed proteins were blotted to poly(vinylidene difluoride) membrane, and biotinylated LDH was detected by alkaline phosphatase-conjugated streptavidin (Vector Laboratories).

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis

    Surface plasmon resonance analysis of biotin-tubulin binding to strepavidin and subsequent dimer dissociation as a result of dilution. (A) The plasmon resonance signal was increased by 954 RU during a 10-min flow of biotin-tubulin in Pi buffer with 12 mM Mg. The almost instantaneous 3000 RU signal change at the start and finish of the flow of the tubulin resulted from a difference in refractive index of the tubulin solution and the buffer. (B) Flow of tubulin- and nucleotide-free buffer resulted in a 445 RU signal decrease; the curve corresponds to a rate constant 14.72 × 10 −5 s −1 . A rate constant equal to 12.35 × 10 −5 s −1 was determined from a Guggenheim plot of the data.

    Journal: Molecular Biology of the Cell

    Article Title: Dissociation of the Tubulin Dimer Is Extremely Slow, Thermodynamically Very Unfavorable, and Reversible in the Absence of an Energy Source

    doi: 10.1091/mbc.E01-10-0089

    Figure Lengend Snippet: Surface plasmon resonance analysis of biotin-tubulin binding to strepavidin and subsequent dimer dissociation as a result of dilution. (A) The plasmon resonance signal was increased by 954 RU during a 10-min flow of biotin-tubulin in Pi buffer with 12 mM Mg. The almost instantaneous 3000 RU signal change at the start and finish of the flow of the tubulin resulted from a difference in refractive index of the tubulin solution and the buffer. (B) Flow of tubulin- and nucleotide-free buffer resulted in a 445 RU signal decrease; the curve corresponds to a rate constant 14.72 × 10 −5 s −1 . A rate constant equal to 12.35 × 10 −5 s −1 was determined from a Guggenheim plot of the data.

    Article Snippet: After three 10-minute washes in PBS, the membrane was incubated for 0.5–16 h with alkaline phosphatase–conjugated streptavidin (Cat no. 21324; Pierce Chemical , Rockford, IL) diluted 46,000-fold in PBS.

    Techniques: SPR Assay, Binding Assay, Flow Cytometry

    Immunoprecipitation and immunoblotting with anti-L* Ab. Multiple 10-μm-thick deparaffinized brain tissue sections from a DA-infected SJL/J mouse (3 days p.i.) were used for protein extraction. The protein extracted from the brain sections of the uninfected SJL/J mouse was used as a negative control, and the protein extracted from BHK-21 cells infected with DA was used as a positive control. L* was immunoprecipitated from the extracted protein by affinity-purified L* Ab bound to the protein G and immobilized by cross-linker. Immnoprecipitated proteins were separated on an SDS-polyacrylamide gel. The bound Ig (anti-L* Ab) was detected with biotinylated secondary Abs and alkaline phosphatase-conjugated streptavidin using BCIP/NBT. Lanes: 1, SJL/J mouse infected with DA; 2, uninfected SJL/J mouse; 3, BHK-21 cells infected with DA. The arrow indicates the immunoprecipitated L*.

    Journal: Journal of Virology

    Article Title: Epitope-Tagged L* Protein of Theiler's Murine Encephalomyelitis Virus Is Expressed in the Central Nervous System in the Acute Phase of Infection

    doi: 10.1128/JVI.76.24.13049-13054.2002

    Figure Lengend Snippet: Immunoprecipitation and immunoblotting with anti-L* Ab. Multiple 10-μm-thick deparaffinized brain tissue sections from a DA-infected SJL/J mouse (3 days p.i.) were used for protein extraction. The protein extracted from the brain sections of the uninfected SJL/J mouse was used as a negative control, and the protein extracted from BHK-21 cells infected with DA was used as a positive control. L* was immunoprecipitated from the extracted protein by affinity-purified L* Ab bound to the protein G and immobilized by cross-linker. Immnoprecipitated proteins were separated on an SDS-polyacrylamide gel. The bound Ig (anti-L* Ab) was detected with biotinylated secondary Abs and alkaline phosphatase-conjugated streptavidin using BCIP/NBT. Lanes: 1, SJL/J mouse infected with DA; 2, uninfected SJL/J mouse; 3, BHK-21 cells infected with DA. The arrow indicates the immunoprecipitated L*.

    Article Snippet: To examine the enhancement of L* detection with anti-FLAG monoclonal Ab (MAb) in vitro, extracted proteins from BHK-21 cells infected with DA, DA/FLAGL*, or DA/3xFLAGL* were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions on 15% acrylamide gels and immunoblotting was performed by using alkaline phosphatase-conjugated streptavidin.

    Techniques: Immunoprecipitation, Infection, Protein Extraction, Negative Control, Positive Control, Affinity Purification

    Immunoblotting with anti-L* Ab (A), anti-FLAG MAb (B), and anti-TMEV VP1 MAb (C). Proteins extracted from BHK-21 cells infected with DA, DA/FLAGL*, and DA/3xFLAGL* were separated on 15% SDS-polyacrylamide gels. Bound Ig was detected with biotinylated secondary Abs and alkaline phosphatase-conjugated streptavidin using BCIP/NBT. Molecular size markers are indicated at the left. Lanes: 1, DA; 2, DA/FLAGL*; 3, DA/3xFLAGL*.

    Journal: Journal of Virology

    Article Title: Epitope-Tagged L* Protein of Theiler's Murine Encephalomyelitis Virus Is Expressed in the Central Nervous System in the Acute Phase of Infection

    doi: 10.1128/JVI.76.24.13049-13054.2002

    Figure Lengend Snippet: Immunoblotting with anti-L* Ab (A), anti-FLAG MAb (B), and anti-TMEV VP1 MAb (C). Proteins extracted from BHK-21 cells infected with DA, DA/FLAGL*, and DA/3xFLAGL* were separated on 15% SDS-polyacrylamide gels. Bound Ig was detected with biotinylated secondary Abs and alkaline phosphatase-conjugated streptavidin using BCIP/NBT. Molecular size markers are indicated at the left. Lanes: 1, DA; 2, DA/FLAGL*; 3, DA/3xFLAGL*.

    Article Snippet: To examine the enhancement of L* detection with anti-FLAG monoclonal Ab (MAb) in vitro, extracted proteins from BHK-21 cells infected with DA, DA/FLAGL*, or DA/3xFLAGL* were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under reducing conditions on 15% acrylamide gels and immunoblotting was performed by using alkaline phosphatase-conjugated streptavidin.

    Techniques: Infection

    Several HLA-E-binding Mtb peptides can bind to Qa-1. Qa-1 transfected HeLa cells were incubated with 0.5 μM biotinylated-Qdm and either 5 or 10 μM of unlabeled competing peptide. Competition for binding to Qa-1 was determined via flow cytometry of streptavidin-APC staining. (A) Representative histograms of concentration-dependent peptide binding to Qa-1 for Qdm (positive control), OT-1 (negative control), and Mtb peptides P55 P68. (B) Relative peptide binding for 17 Mtb peptides, determined by taking the difference in streptavidin-APC MFI between bio-Qdm alone and bio-Qdm + 10 μM test peptide, normalized to the inhibition of bio-Qdm binding by Qdm. n = 2–4 for each peptide.

    Journal: PLoS Pathogens

    Article Title: MHC Ib molecule Qa-1 presents Mycobacterium tuberculosis peptide antigens to CD8+ T cells and contributes to protection against infection

    doi: 10.1371/journal.ppat.1006384

    Figure Lengend Snippet: Several HLA-E-binding Mtb peptides can bind to Qa-1. Qa-1 transfected HeLa cells were incubated with 0.5 μM biotinylated-Qdm and either 5 or 10 μM of unlabeled competing peptide. Competition for binding to Qa-1 was determined via flow cytometry of streptavidin-APC staining. (A) Representative histograms of concentration-dependent peptide binding to Qa-1 for Qdm (positive control), OT-1 (negative control), and Mtb peptides P55 P68. (B) Relative peptide binding for 17 Mtb peptides, determined by taking the difference in streptavidin-APC MFI between bio-Qdm alone and bio-Qdm + 10 μM test peptide, normalized to the inhibition of bio-Qdm binding by Qdm. n = 2–4 for each peptide.

    Article Snippet: After 18h incubation at 37°C, plates were washed using PBS/0.05% Tween 20 and developed using biotinylated α-IFN-γ mAb (R4.6A2, eBioscience, San Diego, CA), followed by streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and a BCIP/NBT substrate kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Transfection, Incubation, Flow Cytometry, Cytometry, Staining, Concentration Assay, Positive Control, Negative Control, Inhibition

    Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and streptavidin linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.

    Journal: PLoS ONE

    Article Title: High-Threshold Mechanosensitive Ion Channels Blocked by a Novel Conopeptide Mediate Pressure-Evoked Pain

    doi: 10.1371/journal.pone.0000515

    Figure Lengend Snippet: Biotinylated NMB-1 binds to peripherin-positive DRG neurons. Primary cultures of DRG neurons were stained with biotinylated NMB-1 and streptavidin linked to Cy3. (A,C) or alkaline phosphatase (D). Counter-staining with a monoclonal antibody to peripherin (B) shows in merged figure (C) that nearly all NMB-1 binding neurons express peripherin. Large diameter sensory neurons (E) were negative for NMB-1 binding. No signal was visible when NMB-1 was omitted from the staining procedure.

    Article Snippet: Biotinylated NMB-1 was visualized by incubating cultures in alkaline phosphatase-conjugated streptavidin (Chemicon); alkaline phosphatase colour reaction was carried out using BCIP/NBT kit from Vector Laboratories according to the manufacturer's instructions.

    Techniques: Staining, Binding Assay

    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: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

    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: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

    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: Beads were washed in 5× diluted lysis buffer, and bound proteins were subjected to SDS-PAGE and Western blotting with streptavidin–alkaline phosphatase conjugate (Calbiochem).

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

    Characterisation of ppIgG. (A) Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab) 2 antibody, followed by alkaline phosphatase-coupled streptavidin (see Materials and Methods ). (B) Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, E. coli O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. (C) ppIgG was tested for reactivity against Escherichia coli and Salmonella diarizonae by competitive ELISA as described ( Materials and Methods ). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p

    Journal: PLoS ONE

    Article Title: Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs

    doi: 10.1371/journal.pone.0147373

    Figure Lengend Snippet: Characterisation of ppIgG. (A) Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab) 2 antibody, followed by alkaline phosphatase-coupled streptavidin (see Materials and Methods ). (B) Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, E. coli O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. (C) ppIgG was tested for reactivity against Escherichia coli and Salmonella diarizonae by competitive ELISA as described ( Materials and Methods ). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p

    Article Snippet: Then, membranes were incubated with polyclonal biotinylated rabbit anti-Pig F(ab)2 IgG (LSBio, Copenhagen, Denmark) diluted 1:10.000 in TBS-T for 1 hour followed by 3x10 min washes in TBS-T before the last incubation with alkaline phosphatase-conjugated streptavidin (DAKO, Glostrup, Denmark) 1:3000 in TBS-T (1 hour).

    Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Competitive ELISA, Inhibition

    Biotinylated DNA tether binds streptavidin. (A) Schematic of the double-stranded DNA tether prepared from half lambda DNA that contains functional groups as indicated on both ends of the molecule. (B) A representative force-extension curve for a single DNA tether formed between an anti-digoxigenin coated polystyrene particle and a biotin-coated polystyrene particle. The length of the overstretch transition in this figure measures 5.4 |im, consistent with that of a half-lambda DNA.

    Journal: Proceedings of SPIE--the International Society for Optical Engineering

    Article Title: A method for tethering single viral particles for virus-cell interaction studies with optical tweezers

    doi: 10.1117/12.2500050

    Figure Lengend Snippet: Biotinylated DNA tether binds streptavidin. (A) Schematic of the double-stranded DNA tether prepared from half lambda DNA that contains functional groups as indicated on both ends of the molecule. (B) A representative force-extension curve for a single DNA tether formed between an anti-digoxigenin coated polystyrene particle and a biotin-coated polystyrene particle. The length of the overstretch transition in this figure measures 5.4 |im, consistent with that of a half-lambda DNA.

    Article Snippet: Streptavidin-alkaline phosphatase conjugate (Invitrogen, Catalog #43–4322) at a concentration of 1.5 ug/mL was used to detect biotinylated protein.

    Techniques: Lambda DNA Preparation, Functional Assay

    Metabolic biotinylation of individual HIV-1 virions. (A) Western blotting of virion preparations with varied inputs of TfR (indicated in the figure) and BirA expression plasmid detected with streptavidin-alkaline phosphatase conjugate. Loaded samples were diluted to have equal p24 concentrations. Lysate from 293T cells transfected with pTfR-AP-IRES-BirA-ER (pTfR) serves as a positive control. (B) Optical trapping virometry results for individual HIV-1 virions: Left: histogram of Alexa 594 fluorescence, proportional to the number of biotinylated transferrin receptors, in individually trapped virions. Right: histogram of EGFP fluorescence in individually trapped virions. N = 28 virions trapped on the same day.

    Journal: Proceedings of SPIE--the International Society for Optical Engineering

    Article Title: A method for tethering single viral particles for virus-cell interaction studies with optical tweezers

    doi: 10.1117/12.2500050

    Figure Lengend Snippet: Metabolic biotinylation of individual HIV-1 virions. (A) Western blotting of virion preparations with varied inputs of TfR (indicated in the figure) and BirA expression plasmid detected with streptavidin-alkaline phosphatase conjugate. Loaded samples were diluted to have equal p24 concentrations. Lysate from 293T cells transfected with pTfR-AP-IRES-BirA-ER (pTfR) serves as a positive control. (B) Optical trapping virometry results for individual HIV-1 virions: Left: histogram of Alexa 594 fluorescence, proportional to the number of biotinylated transferrin receptors, in individually trapped virions. Right: histogram of EGFP fluorescence in individually trapped virions. N = 28 virions trapped on the same day.

    Article Snippet: Streptavidin-alkaline phosphatase conjugate (Invitrogen, Catalog #43–4322) at a concentration of 1.5 ug/mL was used to detect biotinylated protein.

    Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Positive Control, Fluorescence

    TX-100 solubility of membrane proteins in HA/NA and HAt−/NAt− virus-infected MDCK cells. Influenza virus-infected MDCK cells were pulse-labeled with [ 35 S]-Promix, cell surfaces were biotinylated, cells were extracted with 1% TX-100, and soluble (S) and insoluble (I) fractions were separated by centrifugation. Proteins were immunoprecipitated, biotinylated proteins were recovered with streptavidin-Sepharose beads, and polypeptides were analyzed by SDS-PAGE. (A) Biotinylated HA and NA; (B) biotinylated HA and M 2 ; (C) total HA and M 1 immunoprecipitated using anti-A/Udorn/72 influenza virus serum; (D) quantitation of TX-100-insoluble HA, NA, M 1 , and M 2 proteins (data averaged from two independent experiments).

    Journal: Journal of Virology

    Article Title: Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

    doi:

    Figure Lengend Snippet: TX-100 solubility of membrane proteins in HA/NA and HAt−/NAt− virus-infected MDCK cells. Influenza virus-infected MDCK cells were pulse-labeled with [ 35 S]-Promix, cell surfaces were biotinylated, cells were extracted with 1% TX-100, and soluble (S) and insoluble (I) fractions were separated by centrifugation. Proteins were immunoprecipitated, biotinylated proteins were recovered with streptavidin-Sepharose beads, and polypeptides were analyzed by SDS-PAGE. (A) Biotinylated HA and NA; (B) biotinylated HA and M 2 ; (C) total HA and M 1 immunoprecipitated using anti-A/Udorn/72 influenza virus serum; (D) quantitation of TX-100-insoluble HA, NA, M 1 , and M 2 proteins (data averaged from two independent experiments).

    Article Snippet: The separated proteins were blotted to polyvinylidene difluoride (PVDF) membranes ( ); the surface-biotinylated proteins were detected using alkaline phosphatase-conjugated streptavidin and visualized with Vistra ECF (enhanced chemifluorescence) substrate (Amersham Pharmacia Biotech).

    Techniques: Solubility, HAT Assay, Infection, Labeling, Centrifugation, Immunoprecipitation, SDS Page, Quantitation Assay

    Reduced DIGs association of HAt− and NAt− in virus-infected MDCK cells. HA/NA or HAt−/NAt− virus-infected MDCK cells were pulse-labeled with [ 35 S]-Promix, surface biotinylated, and extracted with TX-100. The lysate was then loaded at the bottom of a flotation sucrose density gradient and subjected to equilibrium centrifugation. The gradient was fractionated from the top, fractions were immunoprecipitated, biotinylated HA or NA was recovered by using streptavidin-Sepharose beads, and biotinylated polypeptides were analyzed by SDS-PAGE. The percentages of DIG-associated (top five fractions) and non-DIG-associated (bottom five fractions) proteins are indicated beneath the gels.

    Journal: Journal of Virology

    Article Title: Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

    doi:

    Figure Lengend Snippet: Reduced DIGs association of HAt− and NAt− in virus-infected MDCK cells. HA/NA or HAt−/NAt− virus-infected MDCK cells were pulse-labeled with [ 35 S]-Promix, surface biotinylated, and extracted with TX-100. The lysate was then loaded at the bottom of a flotation sucrose density gradient and subjected to equilibrium centrifugation. The gradient was fractionated from the top, fractions were immunoprecipitated, biotinylated HA or NA was recovered by using streptavidin-Sepharose beads, and biotinylated polypeptides were analyzed by SDS-PAGE. The percentages of DIG-associated (top five fractions) and non-DIG-associated (bottom five fractions) proteins are indicated beneath the gels.

    Article Snippet: The separated proteins were blotted to polyvinylidene difluoride (PVDF) membranes ( ); the surface-biotinylated proteins were detected using alkaline phosphatase-conjugated streptavidin and visualized with Vistra ECF (enhanced chemifluorescence) substrate (Amersham Pharmacia Biotech).

    Techniques: HAT Assay, Infection, Labeling, Centrifugation, Immunoprecipitation, SDS Page

    TX-100 solubility of membrane proteins in BHK cells infected with HA/NA, HAt−/NA, HA/NAt−, and HAt−/NAt−. Virus-infected or plasmid-transfected BHK cells were pulse-labeled with [ 35 S]-Promix, cell surfaces were biotinylated and extracted with 1% TX-100, and soluble (S) and insoluble (I) fractions were separated by centrifugation. Proteins were immunoprecipitated, surface biotinylated proteins were recovered with streptavidin-Sepharose beads, and polypeptides were analyzed by SDS-PAGE. (A) Surface HA, NA, and M 2 in virus-infected BHK cells; (B) surface HA, NA, and M 2 in plasmid-transfected BHK cells; (C) surface G protein from VSV-infected (VSV Gi) and VSV G cDNA-transfected (VSV Gt) BHK cells; (D) quantification of TX-100-insoluble HA, NA, and M 2 proteins in virus-infected BHK cells; (E) quantification of TX-100-insoluble HA, NA, M 2 , and G proteins expressed from cDNAs. Panels D and E represent the average data from two independent experiments.

    Journal: Journal of Virology

    Article Title: Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

    doi:

    Figure Lengend Snippet: TX-100 solubility of membrane proteins in BHK cells infected with HA/NA, HAt−/NA, HA/NAt−, and HAt−/NAt−. Virus-infected or plasmid-transfected BHK cells were pulse-labeled with [ 35 S]-Promix, cell surfaces were biotinylated and extracted with 1% TX-100, and soluble (S) and insoluble (I) fractions were separated by centrifugation. Proteins were immunoprecipitated, surface biotinylated proteins were recovered with streptavidin-Sepharose beads, and polypeptides were analyzed by SDS-PAGE. (A) Surface HA, NA, and M 2 in virus-infected BHK cells; (B) surface HA, NA, and M 2 in plasmid-transfected BHK cells; (C) surface G protein from VSV-infected (VSV Gi) and VSV G cDNA-transfected (VSV Gt) BHK cells; (D) quantification of TX-100-insoluble HA, NA, and M 2 proteins in virus-infected BHK cells; (E) quantification of TX-100-insoluble HA, NA, M 2 , and G proteins expressed from cDNAs. Panels D and E represent the average data from two independent experiments.

    Article Snippet: The separated proteins were blotted to polyvinylidene difluoride (PVDF) membranes ( ); the surface-biotinylated proteins were detected using alkaline phosphatase-conjugated streptavidin and visualized with Vistra ECF (enhanced chemifluorescence) substrate (Amersham Pharmacia Biotech).

    Techniques: Solubility, Infection, HAT Assay, Plasmid Preparation, Transfection, Labeling, Centrifugation, Immunoprecipitation, SDS Page

    Detection of in vitro bound biotinylated ICPs on A. gemmatalis guts tissue: Cry1Aa (a), Cry1Ac (b), and Cry1Ba (c). Negative control (d) when tissue sections were incubated with the biotinylated ICPs (e.g., Cry1Aa) and omission of AP-conjugated streptavidin or when tissue section were incubated with the AP-conjugated streptavidin and omission of biotinylated ICPs. Light micrograph obtained with Nomarski differential interference contrast illumination.

    Journal: ISRN Microbiology

    Article Title: Receptors and Lethal Effect of Bacillus thuringiensis Insecticidal Crystal Proteins to the Anticarsia gemmatalis (Lepidoptera, Noctuidae)

    doi: 10.1155/2013/940284

    Figure Lengend Snippet: Detection of in vitro bound biotinylated ICPs on A. gemmatalis guts tissue: Cry1Aa (a), Cry1Ac (b), and Cry1Ba (c). Negative control (d) when tissue sections were incubated with the biotinylated ICPs (e.g., Cry1Aa) and omission of AP-conjugated streptavidin or when tissue section were incubated with the AP-conjugated streptavidin and omission of biotinylated ICPs. Light micrograph obtained with Nomarski differential interference contrast illumination.

    Article Snippet: The tissue sections were then incubated with 1.5 to 6 μ g of biotinylated toxins (Cry1Aa, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ea, and Cry2Aa) in the TST-buffer for 1 h. Following a washing step with the TST-buffer, the tissues were covered with 300 μ L of streptavidin-alkaline phosphatase conjugate (Amersham) diluted 1/300 in TST-buffer.

    Techniques: In Vitro, Negative Control, Incubation

    CD72 CTLD specifically binds to Sm/RNP. (A–E) Conventional ELISA. Biotinylated CD72 a and CD72 c CTLD proteins at the indicated concentrations were incubated with ELISA plates coated with the indicated molecules. CD72 CTLD proteins bound to the ELISA plates were detected using alkaline phosphatase–conjugated streptavidin and phosphatase substrate. Data are representative of five independent experiments. (F and G) Competitive ELISA. (F) Binding of biotinylated CD72 c CTLD to Sm/RNP in the presence of various concentrations of unbiotinylated CD72 a and CD72 c CTLD proteins was measured. Representative data of five independent experiments are shown. (G) Mean ± SD of percent inhibition of binding in the presence of 100 µg/ml of the indicated competitors in triplicate is shown. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP

    doi: 10.1084/jem.20160560

    Figure Lengend Snippet: CD72 CTLD specifically binds to Sm/RNP. (A–E) Conventional ELISA. Biotinylated CD72 a and CD72 c CTLD proteins at the indicated concentrations were incubated with ELISA plates coated with the indicated molecules. CD72 CTLD proteins bound to the ELISA plates were detected using alkaline phosphatase–conjugated streptavidin and phosphatase substrate. Data are representative of five independent experiments. (F and G) Competitive ELISA. (F) Binding of biotinylated CD72 c CTLD to Sm/RNP in the presence of various concentrations of unbiotinylated CD72 a and CD72 c CTLD proteins was measured. Representative data of five independent experiments are shown. (G) Mean ± SD of percent inhibition of binding in the presence of 100 µg/ml of the indicated competitors in triplicate is shown. **, P

    Article Snippet: The bound biotinylated CD72c CTLD proteins were detected by alkaline phosphatase–conjugated streptavidin (BD).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Competitive ELISA, Binding Assay, Inhibition

    INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after streptavidin-gold labeling ( e ) or Ni-NTA-gold labeling ( f ).

    Journal: Nature structural & molecular biology

    Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism

    doi: 10.1038/nsmb.3199

    Figure Lengend Snippet: INCA preferentially interacts with caspase-1 CARD . ( a ) Co-expression of His-GFP-ASC CARD and biotinylated INCA showed no significant interaction between INCA and ASC CARD , as indicated by the Western blot. ( b ) Over-stoichiometric amounts of INCA did not inhibit His-GFP-ASC CARD nucleated ASC CARD filament formation. Averages of triplicate experiments were plotted. ( c ) Co-expression of His-GFP-caspase-1 CARD and biotinylated INCA showed interaction between INCA and caspase-1 CARD , as confirmed by the PAGE gel. ( d ) Sub-stoichiometric amounts of INCA inhibited His-GFP-caspase-1 CARD nucleated caspase-1 CARD filament formation. Averages of duplicate experiments were plotted. ( e–f ) Negative-stain electron micrographs of the complex (as shown in panel c at 7 ml) between His-GFP-caspase-1 CARD and biotinylated INCA after streptavidin-gold labeling ( e ) or Ni-NTA-gold labeling ( f ).

    Article Snippet: The presence of biotin was confirmed by Western blot using streptavidin-conjugated alkaline phosphatase (streptavidine-AP, Molecular Probes Cat# S921).

    Techniques: Expressing, Western Blot, Polyacrylamide Gel Electrophoresis, Staining, Labeling

    ICEBERG interacts with caspase-1 CARD by co-mixing. ( a ) Co-expression of His-GFP-caspase-1 CARD and biotinylated ICEBERG. Streptavidin-AP and anti-6×His-tag Western blots were shown to confirm the presence of ICEBERG in complex with His-GFP-caspase-1 CARD after Ni-NTA affinity chromatography. L: Whole cell lysate. S: Soluble fraction. U: Ni-NTA unbound. E: Ni-NTA elute. ( b ) The complex eluted from the void of a Superdex 200 column. ( c–d ) Negative-stain electron micrographs of the complex (as shown in panel b at 7 ml) between His-GFP-caspase-1 CARD and biotinylated ICEBERG after Ni-NTA-gold labeling ( c ) or streptavidin-gold labeling ( d ).

    Journal: Nature structural & molecular biology

    Article Title: Molecular basis of caspase-1 polymerization and its inhibition by a novel capping mechanism

    doi: 10.1038/nsmb.3199

    Figure Lengend Snippet: ICEBERG interacts with caspase-1 CARD by co-mixing. ( a ) Co-expression of His-GFP-caspase-1 CARD and biotinylated ICEBERG. Streptavidin-AP and anti-6×His-tag Western blots were shown to confirm the presence of ICEBERG in complex with His-GFP-caspase-1 CARD after Ni-NTA affinity chromatography. L: Whole cell lysate. S: Soluble fraction. U: Ni-NTA unbound. E: Ni-NTA elute. ( b ) The complex eluted from the void of a Superdex 200 column. ( c–d ) Negative-stain electron micrographs of the complex (as shown in panel b at 7 ml) between His-GFP-caspase-1 CARD and biotinylated ICEBERG after Ni-NTA-gold labeling ( c ) or streptavidin-gold labeling ( d ).

    Article Snippet: The presence of biotin was confirmed by Western blot using streptavidin-conjugated alkaline phosphatase (streptavidine-AP, Molecular Probes Cat# S921).

    Techniques: Expressing, Western Blot, Affinity Chromatography, Staining, Labeling

    In vitro binding of PABP to coronavirus 3′ UTR RNAs. In vitro-translated luciferase or PABP was incubated with 1 μg of biotinylated BCV3′UTR RNAs containing poly(A) tails of 1, 5, 10, or 68 A residues (A) or MHV3′UTR RNAs containing poly(A) tails of 0, 5, 10, or > 50 A residues (B). Immobilized streptavidin was added to recover biotinylated RNA complexes, and samples were washed to remove any unbound RNA or protein. Samples were analyzed by SDS-PAGE (8% polyacrylamide). M (lanes 1 and 12) denotes marker and corresponds to the input amount of radiolabeled luciferase (lane 1) or PABP (lane 12) in each reaction. Lanes 2 and 7 represent the level of background protein binding in the absence of biotinylated RNA for luciferase (lane 2) and PABP (lane 7). (A) Lanes 3 to 6, luciferase recovered from interaction with BCV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with BCV3′UTR RNAs. (B) Lanes 3 to 6, luciferase recovered from interaction with MHV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with MHV3′UTR RNAs.

    Journal: Journal of Virology

    Article Title: Host Protein Interactions with the 3? End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

    doi:

    Figure Lengend Snippet: In vitro binding of PABP to coronavirus 3′ UTR RNAs. In vitro-translated luciferase or PABP was incubated with 1 μg of biotinylated BCV3′UTR RNAs containing poly(A) tails of 1, 5, 10, or 68 A residues (A) or MHV3′UTR RNAs containing poly(A) tails of 0, 5, 10, or > 50 A residues (B). Immobilized streptavidin was added to recover biotinylated RNA complexes, and samples were washed to remove any unbound RNA or protein. Samples were analyzed by SDS-PAGE (8% polyacrylamide). M (lanes 1 and 12) denotes marker and corresponds to the input amount of radiolabeled luciferase (lane 1) or PABP (lane 12) in each reaction. Lanes 2 and 7 represent the level of background protein binding in the absence of biotinylated RNA for luciferase (lane 2) and PABP (lane 7). (A) Lanes 3 to 6, luciferase recovered from interaction with BCV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with BCV3′UTR RNAs. (B) Lanes 3 to 6, luciferase recovered from interaction with MHV3′UTR RNAs; lanes 8 to 11, PABP recovered from interaction with MHV3′UTR RNAs.

    Article Snippet: One half was processed as described for protein detection, and the other half was incubated with streptavidin conjugated to alkaline phosphatase (Zymed) for 15 min at room temperature in 200 μl of 1× binding buffer.

    Techniques: In Vitro, Binding Assay, Luciferase, Incubation, SDS Page, Marker, Protein Binding