horseradish peroxidase streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher horseradish peroxidase streptavidin
    Horseradish Peroxidase Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase streptavidin/product/Thermo Fisher
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase streptavidin - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Electrophoresis:

    Article Title: TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus
    Article Snippet: After electrophoresis and transfer onto membranes, the samples were incubated with rabbit anti-Ro52 IgG (2 µg/ml; Abcam). .. Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color development.

    Immunohistochemistry:

    Article Title: Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study
    Article Snippet: Paragraph title: Immunohistochemistry ... Histostain SP kit containing serum blocking solution, a biotinylated secondary antibody, a horseradish peroxidase streptavidin and a substrate chromogen mixture was purchased (95–9643, LAB-SA system, Zymed Laboratories Inc, San Francisco, CA, USA).

    Amplification:

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: .. The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA). .. The biotin-labelled bpifcl and DIG-labelled kiss2 riboprobes were mixed in a cocktail for the hybridisation.

    Incubation:

    Article Title: Impairment of the humoral and CD4+ T cell responses in HTLV-1-infected individuals immunized with tetanus toxoid
    Article Snippet: Goat anti-human IgG alkaline phosphatase conjugate (Sigma Chemicals, St. Louis, MO, USA) was diluted (1:1,000) in PBS pH 7.2 supplemented with 0.05% Tween 20 and incubated for 1 h at 37°C, and 1 mg/mL p -nitrophenyl phosphate (pNPP, Sigma Chemicals, St. Louis, MO, USA) was used to reveal the reaction. .. Horseradish-peroxidase streptavidin (Invitrogen, Carlsbad, CA, USA) was added for 30 min at room temperature (RT).

    Article Title: Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study
    Article Snippet: They were incubated with the following primary antibodies for 30 minutes: Caspase-3 antibody, primary antisera diluted in antibody diluents (1:500) (TA-125-UD, Lab vision, Goteborg, Sweden). .. Histostain SP kit containing serum blocking solution, a biotinylated secondary antibody, a horseradish peroxidase streptavidin and a substrate chromogen mixture was purchased (95–9643, LAB-SA system, Zymed Laboratories Inc, San Francisco, CA, USA).

    Article Title: TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus
    Article Snippet: After electrophoresis and transfer onto membranes, the samples were incubated with rabbit anti-Ro52 IgG (2 µg/ml; Abcam). .. Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color development.

    Article Title: Influence of Type I Collagen Surface Density on Fibroblast Spreading, Motility, and Contractility
    Article Snippet: .. After rinsing thoroughly with 0.1% Tween-20 in 1× phosphate buffered saline three times, the substrata are incubated with 1.5 mL horseradish peroxidase-streptavidin (Pierce Biotechnology, Rockford, IL) at a dilution of 1:20,000 in blocking buffer for 30 min at 37°C and then thoroughly washed again. .. Tetramethylbenzidine (Pierce Biotechnology) is then added to the substrata and the color is allowed to develop at room temperature for 10 min.

    Article Title: Induction and suppression of allergic diarrhea and systemic anaphylaxis in a mouse model of food allergy
    Article Snippet: .. After incubation for 60 minutes (min), bound IgA or IgE were detected with biotin-anti-mouse IgAmAb (BD Bioscience), or biotin-rat IgG2a anti-mouse IgE mAb (RIE4), respectively, followed sequentially by horseradish peroxidase-streptavidin and SuperSignal ELISA substrate (Pierce Biotechnology). .. The significance of differences in temperature and Ig concentrations between groups of mice was compared with the Mann-Whitney t test.

    Article Title: Regulation of CD154-induced interleukin-12 production in synovial fluid macrophages
    Article Snippet: After three washes, the samples and standards (IL-12p70 or IL-12p40), resuspended in 0.1% BSA, 0.05% Tween 20 in Tris-buffered saline solution, pH7.3, were added and incubated for 2 hours at room temperature. .. The plates were again washed three times and horseradish peroxidase:streptavidin (1:20000, 100 μl/well; 43–4323, Zymed Laboratories, San Francisco, CA, USA) was added for 20 minutes.

    Article Title: Proteome-wide modulation of degradation dynamics in response to growth arrest
    Article Snippet: .. After 1 h incubation with AHA, cells were collected and lysed; 50 μL of 1 mg/mL lysate was used for click reaction using Click Chemistry Protein Reaction Buffer Kit (Click Chemistry Tools); 20 μL of click reaction product was used for Western blot and probed by Horseradish peroxidase streptavidin (Thermo Fisher Scientific). ..

    Article Title: Serum, plasma, and dried blood spot high sensitivity C-reactive protein enzyme immunoassay for population research
    Article Snippet: .. After incubation for 2 h at room temperature, plates were washed and 100µL/well of horseradish peroxidase streptavidin (Invitrogen Corporation) diluted 1:6000 in assay buffer was added. .. After incubating 1 h at room temperature, plates were washed again, and developed in citrate buffer (50 mmol citrate, pH 4.0) combined with 0.4 mmol 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich) and 1.6 mmol of hydrogen peroxide (100µL/well).

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA). .. After hybridisation, a peroxidase-conjugated anti-DIG antibody (Roche Diagnostics) diluted 1:500 in a buffer containing 0.1 M Tris (pH 7.4), 0.15 M NaCl and Tween buffer (0.05% Triton X-100) with 1% normal goat serum was applied to each slide for 2 h at room temperature, followed by incubation with Tyramide Signal Amplification Working Solution for 1 min for colour development of kiss2 probes.

    Article Title: Implication of SUMO E3 ligases in nucleotide excision repair
    Article Snippet: .. After washing, either mouse monoclonal anti-CPD or mouse monoclonal anti-(6-4)PP antibodies (Cosmo Bio, Tokyo, Japan) diluted in PBS were distributed to DNA-coated plates, which were then incubated at 37 °C for 30 min. After washing, these plates were incubated with the Biotin-XX F(ab’)2 fragment of anti-mouse IgG (H+L) (Invitrogen Life Technologies) at 37 °C for 30 min, followed by incubation with Horseradish Peroxidase-Streptavidin (Invitrogen Life Technologies). .. Captured CPDs and 6-4PPs were visualized using BD OptEIA™ Substrate Reagents A and B (BD Biosciences, San Jose, CA).

    Fluorescence:

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: Paragraph title: Double-label fluorescence ISH of bpifcl and kiss2 mRNA ... The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA).

    Software:

    Article Title: TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus
    Article Snippet: Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color development. .. The ImageJ1.61u software was used for quantitation of the band intensities, which were then normalized to the β-actin values accordingly.

    Negative Control:

    Article Title: Proteome-wide modulation of degradation dynamics in response to growth arrest
    Article Snippet: Then, media was replaced by RPMI, no methionine media supplemented with 15% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 4 mM l -AHA; 100 μg/mL cycloheximide was used to inhibit protein synthesis as negative control. .. After 1 h incubation with AHA, cells were collected and lysed; 50 μL of 1 mg/mL lysate was used for click reaction using Click Chemistry Protein Reaction Buffer Kit (Click Chemistry Tools); 20 μL of click reaction product was used for Western blot and probed by Horseradish peroxidase streptavidin (Thermo Fisher Scientific).

    Blocking Assay:

    Article Title: Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study
    Article Snippet: .. Histostain SP kit containing serum blocking solution, a biotinylated secondary antibody, a horseradish peroxidase streptavidin and a substrate chromogen mixture was purchased (95–9643, LAB-SA system, Zymed Laboratories Inc, San Francisco, CA, USA). ..

    Article Title: Influence of Type I Collagen Surface Density on Fibroblast Spreading, Motility, and Contractility
    Article Snippet: .. After rinsing thoroughly with 0.1% Tween-20 in 1× phosphate buffered saline three times, the substrata are incubated with 1.5 mL horseradish peroxidase-streptavidin (Pierce Biotechnology, Rockford, IL) at a dilution of 1:20,000 in blocking buffer for 30 min at 37°C and then thoroughly washed again. .. Tetramethylbenzidine (Pierce Biotechnology) is then added to the substrata and the color is allowed to develop at room temperature for 10 min.

    Article Title: Regulation of CD154-induced interleukin-12 production in synovial fluid macrophages
    Article Snippet: Plates (Immulon, Dynatech Laboratories, Chantilly, VA, USA) were prepared by coating with monoclonal capture antibodies (3 μg/ml antihuman IL-12p70 mAb or antihuman IL-12p40 mAb) overnight at room temperature followed by three washes with washing buffer (0.05% Tween 20 in phosphate-buffered saline solution) and then blocking with phosphate-buffered saline solution containing 1% BSA, 5% sucrose, and 0.05% NaN3 for 1 hour. .. The plates were again washed three times and horseradish peroxidase:streptavidin (1:20000, 100 μl/well; 43–4323, Zymed Laboratories, San Francisco, CA, USA) was added for 20 minutes.

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA). .. After blocking with 3% H2 O2 in a 0.1 M Tris-HCl (pH 7.4) and 0.15 M NaCl buffer for 30 min at room temperature, a horseradish peroxidase-conjugated streptavidin antibody diluted 1:100 in the 0.1 M Tris-HCl (pH 7.4) and 0.15 M NaCl buffer buffer with 2% bovine serum albumin was applied to each slide overnight at 4 °C.

    Article Title: Implication of SUMO E3 ligases in nucleotide excision repair
    Article Snippet: Plates were then washed with PBS containing 0.05 % Tween-20, followed by blocking with 2 % FBS. .. After washing, either mouse monoclonal anti-CPD or mouse monoclonal anti-(6-4)PP antibodies (Cosmo Bio, Tokyo, Japan) diluted in PBS were distributed to DNA-coated plates, which were then incubated at 37 °C for 30 min. After washing, these plates were incubated with the Biotin-XX F(ab’)2 fragment of anti-mouse IgG (H+L) (Invitrogen Life Technologies) at 37 °C for 30 min, followed by incubation with Horseradish Peroxidase-Streptavidin (Invitrogen Life Technologies).

    Mouse Assay:

    Article Title: Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study
    Article Snippet: Sections were rinsed twice in PBS Tween 20 for 2 minutes and blocked with 5% normal mice serum for 30 minutes at room temperature. .. Histostain SP kit containing serum blocking solution, a biotinylated secondary antibody, a horseradish peroxidase streptavidin and a substrate chromogen mixture was purchased (95–9643, LAB-SA system, Zymed Laboratories Inc, San Francisco, CA, USA).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Influence of Type I Collagen Surface Density on Fibroblast Spreading, Motility, and Contractility
    Article Snippet: After rinsing thoroughly with 0.1% Tween-20 in 1× phosphate buffered saline three times, the substrata are incubated with 1.5 mL horseradish peroxidase-streptavidin (Pierce Biotechnology, Rockford, IL) at a dilution of 1:20,000 in blocking buffer for 30 min at 37°C and then thoroughly washed again. .. The reaction is stopped with 1.5 mL 1M H2 SO4 , and then 200 μ L of the mixture is transferred to a 96-well flat-bottomed ELISA plate (Corning, Corning, NY).

    Article Title: Induction and suppression of allergic diarrhea and systemic anaphylaxis in a mouse model of food allergy
    Article Snippet: .. After incubation for 60 minutes (min), bound IgA or IgE were detected with biotin-anti-mouse IgAmAb (BD Bioscience), or biotin-rat IgG2a anti-mouse IgE mAb (RIE4), respectively, followed sequentially by horseradish peroxidase-streptavidin and SuperSignal ELISA substrate (Pierce Biotechnology). .. The significance of differences in temperature and Ig concentrations between groups of mice was compared with the Mann-Whitney t test.

    Article Title: Implication of SUMO E3 ligases in nucleotide excision repair
    Article Snippet: Paragraph title: ELISA ... After washing, either mouse monoclonal anti-CPD or mouse monoclonal anti-(6-4)PP antibodies (Cosmo Bio, Tokyo, Japan) diluted in PBS were distributed to DNA-coated plates, which were then incubated at 37 °C for 30 min. After washing, these plates were incubated with the Biotin-XX F(ab’)2 fragment of anti-mouse IgG (H+L) (Invitrogen Life Technologies) at 37 °C for 30 min, followed by incubation with Horseradish Peroxidase-Streptavidin (Invitrogen Life Technologies).

    Quantitation Assay:

    Article Title: TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus
    Article Snippet: Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color development. .. The ImageJ1.61u software was used for quantitation of the band intensities, which were then normalized to the β-actin values accordingly.

    Activity Assay:

    Article Title: Postnatal changes in the development of rat submandibular glands in offspring of diabetic mothers: Biochemical, histological and ultrastructural study
    Article Snippet: The peroxidase activity was demonstrated by AEC (3-amino-9-ethyl carbazole) substrate kit (TA- 004HAC, Lab vision, Goteborg, Sweden). .. Histostain SP kit containing serum blocking solution, a biotinylated secondary antibody, a horseradish peroxidase streptavidin and a substrate chromogen mixture was purchased (95–9643, LAB-SA system, Zymed Laboratories Inc, San Francisco, CA, USA).

    Labeling:

    Article Title: Proteome-wide modulation of degradation dynamics in response to growth arrest
    Article Snippet: Paragraph title: l -AHA Labeling. ... After 1 h incubation with AHA, cells were collected and lysed; 50 μL of 1 mg/mL lysate was used for click reaction using Click Chemistry Protein Reaction Buffer Kit (Click Chemistry Tools); 20 μL of click reaction product was used for Western blot and probed by Horseradish peroxidase streptavidin (Thermo Fisher Scientific).

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: Double-label fluorescence ISH of bpifcl and kiss2 mRNA Riboprobes for bpifcl and kiss2 mRNA were labelled with biotin and DIG, respectively, using MAXIscript with either biotin or DIG RNA Labeling Mix (Roche Diagnostics). .. The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA).

    Western Blot:

    Article Title: TWEAK/Fn14 Activation Participates in Ro52-Mediated Photosensitization in Cutaneous Lupus Erythematosus
    Article Snippet: Paragraph title: Western Blotting ... Horseradish peroxidase-streptavidin and an ECL kit (Thermo Scientific, Waltham, MA, USA) were used for color development.

    Article Title: Proteome-wide modulation of degradation dynamics in response to growth arrest
    Article Snippet: .. After 1 h incubation with AHA, cells were collected and lysed; 50 μL of 1 mg/mL lysate was used for click reaction using Click Chemistry Protein Reaction Buffer Kit (Click Chemistry Tools); 20 μL of click reaction product was used for Western blot and probed by Horseradish peroxidase streptavidin (Thermo Fisher Scientific). ..

    Purification:

    Article Title: Induction and suppression of allergic diarrhea and systemic anaphylaxis in a mouse model of food allergy
    Article Snippet: IgA and IgE concentrations were determined by sandwich ELISAs in which microtiter plate wells were coated with anti-mouse IgA mAb (BD Bioscience, Franklin Lakes, NJ) or rat IgGa anti-mouse IgE mAb (EM-95) followed by sample and purified IgA or IgE standard (BD Biosciences). .. After incubation for 60 minutes (min), bound IgA or IgE were detected with biotin-anti-mouse IgAmAb (BD Bioscience), or biotin-rat IgG2a anti-mouse IgE mAb (RIE4), respectively, followed sequentially by horseradish peroxidase-streptavidin and SuperSignal ELISA substrate (Pierce Biotechnology).

    Article Title: Implication of SUMO E3 ligases in nucleotide excision repair
    Article Snippet: Genomic DNAs were purified from UV-irradiated cells using MagExtractor-Genome (TOYOBO, Osaka, Japan). .. After washing, either mouse monoclonal anti-CPD or mouse monoclonal anti-(6-4)PP antibodies (Cosmo Bio, Tokyo, Japan) diluted in PBS were distributed to DNA-coated plates, which were then incubated at 37 °C for 30 min. After washing, these plates were incubated with the Biotin-XX F(ab’)2 fragment of anti-mouse IgG (H+L) (Invitrogen Life Technologies) at 37 °C for 30 min, followed by incubation with Horseradish Peroxidase-Streptavidin (Invitrogen Life Technologies).

    In Situ Hybridization:

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: Paragraph title: Double-label fluorescence ISH of bpifcl and kiss2 mRNA ... The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA).

    Hybridization:

    Article Title: Bpifcl modulates kiss2 expression under the influence of 11-ketotestosterone in female zebrafish
    Article Snippet: The biotin-labelled bpifcl probe was detected using horseradish peroxidase-streptavidin and AlexaFluor 594 Tyramide (Invitrogen), whereas the DIG-labelled kiss2 probe was detected using a Tyramide Signal Amplification Plus kit (PerkinElmer/NEN Life Science Products, MA, USA). .. The biotin-labelled bpifcl and DIG-labelled kiss2 riboprobes were mixed in a cocktail for the hybridisation.

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    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, 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/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 162 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp - by Bioz Stars, 2020-03
    99/100 stars
      Buy from Supplier

    Image Search Results


    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Journal: BMC Cell Biology

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    doi: 10.1186/s12860-014-0045-1

    Figure Lengend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Article Snippet: Each diluted standard was incubated with primary antibody coated on the well and streptavidin-HRP and OD value for each sample was detected by ELISA.

    Techniques: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Journal: The Journal of Biological Chemistry

    Article Title: PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *

    doi: 10.1074/jbc.M113.541250

    Figure Lengend Snippet: Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Article Snippet: For the second experiment, 4 μl of V-ATPase (4 μg) was mixed with 3 μl of biotin-PA1b (3 μg) and 3 μl of streptavidin-HRP (15 μg), made up to 60 μl using V-ATPase buffer and incubated for 30 min. Mg·ATP was from a stock solution of 100 mm at pH 7.5 to a final concentration of 5 mm , and the mixture was incubated at room temperature for 5 min to allow for complete turnover.

    Techniques: Electron Microscopy, Generated, Produced

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Journal: Veterinary Research

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    doi: 10.1051/vetres/2010048

    Figure Lengend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Article Snippet: The expression of GFP-fused proteins in these clones was confirmed by Western blot using a biotin-conjugated goat anti-GFP polyclonal antibody and streptavidin-HRP.

    Techniques: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing