peroxidase conjugated streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher peroxidase conjugated streptavidin
    In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by <t>streptavidin-HRP,</t> chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.
    Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab"

    Article Title: Aptamers as quality control tool for production, storage and biosimilarity of the anti-CD20 biopharmaceutical rituximab

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37624-1

    In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by streptavidin-HRP, chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.
    Figure Legend Snippet: In vitro selection and binding of aptamers. ( a ) Schematic representation of the SELEX process used within this study. ( b ) Schematic illustration of the enzyme linked apta-sorbent assay (ELASA) setup. ( c ) Folded biotinylated aptamers (RA1-RA9, c = 500 nM) were tested for rituximab binding. Bound aptamers were detected by streptavidin-HRP, chemiluminescence ELISA substrate was used for detection and luminescence was measured. Measurements were performed in six replicates; means and standard errors of the mean are given.

    Techniques Used: In Vitro, Selection, Binding Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through theBRCA2 Promoter *"

    Article Title: Poly(ADP-ribose) Polymerase-1 Down-regulates BRCA2 Expression through theBRCA2 Promoter *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803693200

    Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.
    Figure Legend Snippet: Parp-1 binds to the BRCA2 promoter. A , MCF-7 cell nuclear extracts were mixed with a biotinylated WT probe and a mutant probe ( M ). The DNA-protein complex was isolated with streptavidin-labeled magnetic beads. The magnetic bead column was washed and eluates were collected. Eluted fractions were separated on 10% acrylamide gels and visualized by silver staining. The 120-kDa protein band indicates a prominent DNA-protein complex. BSA was used as a control, and its position is indicated by a black arrow . The experiment was carried out three times. E1, E2, E3 (WT probe eluates); M , mutant probe eluate. B , mass spectrometry of the ∼120-kDa protein. C , EMSAs and antibody super-shifts were performed in MCF-7 nuclear extracts using WT, mutant probes, and Parp-1 antibody. D , EMSAs were carried out in MCF-7 nuclear extracts using the end-streptavidin blocked-biotin-labeled WT and mutant probes. E , luciferase activity of Del-9 in MCF-7 cells treated with 3-AB/NU1025, two Parp-1 inhibitors, or DMSO for 12 h.

    Techniques Used: Mutagenesis, Isolation, Labeling, Magnetic Beads, Silver Staining, Mass Spectrometry, Luciferase, Activity Assay

    3) Product Images from "Use of a ?1 Integrin-deficient Human T Cell to Identify ?1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function"

    Article Title: Use of a ?1 Integrin-deficient Human T Cell to Identify ?1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

    Journal: Molecular Biology of the Cell

    doi:

    The A1 mutant does not express β1 integrin protein. Jurkat cells and the A1 mutant were biotinylated and lysed in 1% Triton detergent, and lysates were subjected to immunoprecipitation (IP) with goat anti-mouse Sepharose beads conjugated with the β1-specific mAb P4C10 or the β2-specific mAb TS1/22. IPs were washed, run on 5% acrylamide gel, and transferred to nitrocellulose. Protein was detected by incubating the membrane with Streptavidin-HRP and visualized by chemiluminescence.
    Figure Legend Snippet: The A1 mutant does not express β1 integrin protein. Jurkat cells and the A1 mutant were biotinylated and lysed in 1% Triton detergent, and lysates were subjected to immunoprecipitation (IP) with goat anti-mouse Sepharose beads conjugated with the β1-specific mAb P4C10 or the β2-specific mAb TS1/22. IPs were washed, run on 5% acrylamide gel, and transferred to nitrocellulose. Protein was detected by incubating the membrane with Streptavidin-HRP and visualized by chemiluminescence.

    Techniques Used: Mutagenesis, Immunoprecipitation, Acrylamide Gel Assay

    4) Product Images from "Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta"

    Article Title: Development and Validation of an Enzyme-Linked Immunosorbent Assay for the Detection of Binding Anti-Drug Antibodies against Interferon Beta

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2017.00305

    Assay principle. Interferon beta (IFN-β) 1a is non-specifically bound to the surface allowing the presentation of multiple epitopes. A “bridge” is formed by the subsequent addition of a positive serum sample or rabbit antihuman IFN-β and biotin-labeled IFN-β 1a. The latter is detected by HRP-conjugated streptavidin.
    Figure Legend Snippet: Assay principle. Interferon beta (IFN-β) 1a is non-specifically bound to the surface allowing the presentation of multiple epitopes. A “bridge” is formed by the subsequent addition of a positive serum sample or rabbit antihuman IFN-β and biotin-labeled IFN-β 1a. The latter is detected by HRP-conjugated streptavidin.

    Techniques Used: Labeling

    5) Product Images from "Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18"

    Article Title: Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

    Journal: The Journal of nutritional biochemistry

    doi: 10.1016/j.jnutbio.2010.04.001

    HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).
    Figure Legend Snippet: HCS interacts physically with histone H3.2 in HEK293 cells Panel A: Nuclear extracts from HEK293 HCS-GFP cells were precipitated with anti-GFP and probed with anti-histone H3. Nuclear extracts precipitated with non-specific IgG was used as negative control. IgG precipitates (without anti-GFP) were probed with anti-histone H3 (“IgG”); Nuclear extracts without antibody treatment (input), and 0.1 μg of recombinant human histone H3.2 (rH3.2) were used as positive controls. Panel B: Nuclear extracts from HEK293 cells were precipitated with anti-HCS and probed with anti-histone H3 (“HCS pulldown”). As positive control, nuclear extracts collected before treatment with anti-HCS were probed with anti-histone H3 (“Input”); as negative control, protein A precipitates (without anti-HCS) were probed with anti-histone H3 (“Protein A”). Panel C: Purified rHCS was probed with anti-human HCS (lane 1), anti-poly·his tag (lane 2), and coomassie blue (lane 3). Panel D: rHCS was incubated with p67 and cofactors for enzymatic biotinylation; negative controls were generated by omission of individual compounds from reaction mixtures. p67-bound biotin was probed using streptavidin. Panel E: Preincubation of H3 with HCS protects H3 against proteolysis by trypsin in limited proteolysis assays. Left = recombinant histone H3.2 alone; middle = H3.2 pre-incubated with HCS-GST; right = H3.2 pre-incubated with GST alone. H3 was probed with coomassie blue (top panel) and anti-histone H3.2 (bottom panel).

    Techniques Used: Negative Control, Recombinant, Positive Control, Purification, Incubation, Generated

    Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.
    Figure Legend Snippet: Recombinant human HCS catalyzes biotinylation of histone H3.2 Panel A: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for up to 12 h; negative controls were generated by omission of rHCS and H3.2. Samples were collected at timed intervals and histone-bound biotin was probed using anti-biotin. Equal loading of histone H3.2 was confirmed by staining with Coomassie blue. Panel B: rHCS was incubated with recombinant human histone H3.2 and cofactors for enzymatic biotinylation for 12 h; negative controls were generated by omission of rHCS and H3.2. Histone-bound biotin was probed using streptavidin. Staining with Coomassie blue was used as loading control. Panel C: Same as panel B, but a synthetic peptide representing amino acids 1 to 25 (N 1–25 ) in human histone H3.2 was used as substrate. Negative controls were generated by omission of rHCS and peptide.

    Techniques Used: Recombinant, Incubation, Generated, Staining

    6) Product Images from "The α2β1 integrin mediates the malignant phenotype on type I collagen in pancreatic cancer cell lines"

    Article Title: The α2β1 integrin mediates the malignant phenotype on type I collagen in pancreatic cancer cell lines

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6603088

    Integrin, E-cadherin, and β -catenin expression in pancreatic cancer cell lines. Immunoprecipitations were conducted using 4 μ g anti-integrin antibodies and 400 μ g cell-surface-biotinylated extracts from pancreatic cancer cell lines as described in Materials and Methods. Immunoprecipitates were separated on 12% Nu–PAGE gels under nonreducing conditions, and protein bands were visualised using streptavidin–HRP and chemiluminescence. Autoradiograms for each particular integrin subunit or integrin heterodimer for each cell line are indicated in the right-hand margin.
    Figure Legend Snippet: Integrin, E-cadherin, and β -catenin expression in pancreatic cancer cell lines. Immunoprecipitations were conducted using 4 μ g anti-integrin antibodies and 400 μ g cell-surface-biotinylated extracts from pancreatic cancer cell lines as described in Materials and Methods. Immunoprecipitates were separated on 12% Nu–PAGE gels under nonreducing conditions, and protein bands were visualised using streptavidin–HRP and chemiluminescence. Autoradiograms for each particular integrin subunit or integrin heterodimer for each cell line are indicated in the right-hand margin.

    Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis

    7) Product Images from "Naturally Occurring Glucokinase Mutations Are Associated with Defects in Posttranslational S-Nitrosylation"

    Article Title: Naturally Occurring Glucokinase Mutations Are Associated with Defects in Posttranslational S-Nitrosylation

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2009-0138

    ). S-nitrosylated proteins from cell lysates were labeled with biotin, and GCK-mVenus proteins were immunoprecipitated. Precipitates were analyzed by Western blot using anti-GFP antibodies ( top ) and streptavidin-horseradish peroxidase ( bottom ) to detect S-nitrosylated proteins. Strept-HRP, Streptavidin-horseradish peroxidase.
    Figure Legend Snippet: ). S-nitrosylated proteins from cell lysates were labeled with biotin, and GCK-mVenus proteins were immunoprecipitated. Precipitates were analyzed by Western blot using anti-GFP antibodies ( top ) and streptavidin-horseradish peroxidase ( bottom ) to detect S-nitrosylated proteins. Strept-HRP, Streptavidin-horseradish peroxidase.

    Techniques Used: Labeling, Immunoprecipitation, Western Blot

    8) Product Images from "An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse"

    Article Title: An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0356

    Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.
    Figure Legend Snippet: Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.

    Techniques Used: Transfection, Immunostaining, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, CTL Assay, MANN-WHITNEY

    9) Product Images from "Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells *"

    Article Title: Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.041152

    Exosome release in acidic and buffered pH conditions. A , exosomes (100 μg) isolated from Mel1 cells culture medium were NHS-biotin-labeled and loaded on a 10–60% continuous sucrose gradient. Eleven fractions were analyzed by Western blotting with horseradish peroxidase-streptavidin. Results indicate a molecules profile with molecular mass ranging between 97 and 21 kDa enriched in fractions 3–6 corresponding to density 1.11–1.17 g/ml. These fractions were also found positive for exosome markers such as Lamp-2, CD81, and Rab 5B by Western blotting. B , Mel1 cells (3 × 10 6 cell/ml) were cultured in buffered (pH 7.4) or acidic (pH 6.0) media. At the indicated days exosomes were isolated, quantified by protein assay, and normalized on 1 × 10 6 viable cells. Points , means ( n = 4); bars , S.D. *, p
    Figure Legend Snippet: Exosome release in acidic and buffered pH conditions. A , exosomes (100 μg) isolated from Mel1 cells culture medium were NHS-biotin-labeled and loaded on a 10–60% continuous sucrose gradient. Eleven fractions were analyzed by Western blotting with horseradish peroxidase-streptavidin. Results indicate a molecules profile with molecular mass ranging between 97 and 21 kDa enriched in fractions 3–6 corresponding to density 1.11–1.17 g/ml. These fractions were also found positive for exosome markers such as Lamp-2, CD81, and Rab 5B by Western blotting. B , Mel1 cells (3 × 10 6 cell/ml) were cultured in buffered (pH 7.4) or acidic (pH 6.0) media. At the indicated days exosomes were isolated, quantified by protein assay, and normalized on 1 × 10 6 viable cells. Points , means ( n = 4); bars , S.D. *, p

    Techniques Used: Isolation, Labeling, Western Blot, Cell Culture

    Role of microenvironmental pH in exosome fusion. A , R18-exoMel1 and R18-exoMel1 ac were mixed with both acidic and buffered Mel1 cells (1 × 10 6 ), and fusion efficiency was tested for 30 min. Points , means ± S.D. FD , fluorescence dequenching. B , NHS-biotin-buffered (pH 7.4) or acidic (pH 6.0) exosomes (25 μg) were incubated for 1 h with untreated or PPI-treated parental cells, then cells were analyzed by streptavidin blotting. Western blotting of nucleolin represents a control for cell protein equal loading. Numbers represent the whole lane densitometry analysis expressed in arbitrary units ( a.u .). Points , means ± S.D. a p
    Figure Legend Snippet: Role of microenvironmental pH in exosome fusion. A , R18-exoMel1 and R18-exoMel1 ac were mixed with both acidic and buffered Mel1 cells (1 × 10 6 ), and fusion efficiency was tested for 30 min. Points , means ± S.D. FD , fluorescence dequenching. B , NHS-biotin-buffered (pH 7.4) or acidic (pH 6.0) exosomes (25 μg) were incubated for 1 h with untreated or PPI-treated parental cells, then cells were analyzed by streptavidin blotting. Western blotting of nucleolin represents a control for cell protein equal loading. Numbers represent the whole lane densitometry analysis expressed in arbitrary units ( a.u .). Points , means ± S.D. a p

    Techniques Used: Fluorescence, Incubation, Western Blot

    10) Product Images from "Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import"

    Article Title: Multiple 40-kDa Heat-Shock Protein Chaperones Function in Tom70-dependent Mitochondrial Import

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-01-0088

    Hsc70-preprotein interactions. (A) Top, Hsc70 was radiolabeled by cell-free translation, incubated with ANT-B immobilized on streptavidin-agarose, and coprecipitated after the indicated times. Reactions contained either no addition, or 20 μM purified
    Figure Legend Snippet: Hsc70-preprotein interactions. (A) Top, Hsc70 was radiolabeled by cell-free translation, incubated with ANT-B immobilized on streptavidin-agarose, and coprecipitated after the indicated times. Reactions contained either no addition, or 20 μM purified

    Techniques Used: Incubation, Purification

    11) Product Images from "Ephrin B1 Is Expressed on Neuroepithelial Cells in Correlation with Neocortical Neurogenesis"

    Article Title: Ephrin B1 Is Expressed on Neuroepithelial Cells in Correlation with Neocortical Neurogenesis

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-08-02726.2001

    Purification of the 25H11 antigen. A, Flow scheme of the purification of the 25H11 antigen from E13.5 mouse telencephali and of its further processing for nanosequencing. For details, see Materials and Methods. B, Aliquots (0.2%) of the eluate from the 25H11-Sepharose ( A ) were analyzed by SDS-PAGE followed by silver staining or were biotinylated and analyzed by SDS-PAGE followed by streptavidin blotting. Arrow , 25H11 antigen. C , C ′, The 25H11 antigen was purified through the 25H11-Sepharose step as described in A , except that only ≈140 E13.5 mouse telencephali were used. An aliquot (25%) of the eluate from the 25H11-Sepharose was PEG-precipitated and biotinylated, and an aliquot of it (80%) was analyzed by two-dimensional PAGE (acidic, right ) followed by immunoblotting with the 25H11 antibody ( C , ≈5 min time ECL exposure). The nitrocellulose was incubated in reducing condition to remove the antibody and reprobed with streptavidin-HRP ( C ′, ≈1 min time ECL exposure).
    Figure Legend Snippet: Purification of the 25H11 antigen. A, Flow scheme of the purification of the 25H11 antigen from E13.5 mouse telencephali and of its further processing for nanosequencing. For details, see Materials and Methods. B, Aliquots (0.2%) of the eluate from the 25H11-Sepharose ( A ) were analyzed by SDS-PAGE followed by silver staining or were biotinylated and analyzed by SDS-PAGE followed by streptavidin blotting. Arrow , 25H11 antigen. C , C ′, The 25H11 antigen was purified through the 25H11-Sepharose step as described in A , except that only ≈140 E13.5 mouse telencephali were used. An aliquot (25%) of the eluate from the 25H11-Sepharose was PEG-precipitated and biotinylated, and an aliquot of it (80%) was analyzed by two-dimensional PAGE (acidic, right ) followed by immunoblotting with the 25H11 antibody ( C , ≈5 min time ECL exposure). The nitrocellulose was incubated in reducing condition to remove the antibody and reprobed with streptavidin-HRP ( C ′, ≈1 min time ECL exposure).

    Techniques Used: Purification, Flow Cytometry, SDS Page, Silver Staining, Polyacrylamide Gel Electrophoresis, Incubation

    12) Product Images from "An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse"

    Article Title: An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-06-0356

    Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.
    Figure Legend Snippet: Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.

    Techniques Used: Transfection, Immunostaining, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, CTL Assay, MANN-WHITNEY

    13) Product Images from "Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models"

    Article Title: Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-46

    Inhibitory effect of purified MB-MoG-3.7 (grey bars), MB-MoG-2.8 (black bars) and mAb CUB7402 (hatched bars) on mouse tTG activity. Elisa plates coated with gliadin, a tTG substrate, are incubated with 0.2 mM 5-(biotinamido)pentylamine and 0.25 μg of mouse tTG, with increasing amounts of purified miniantibody or mAb. The incorporation of 5-(biotinamido)pentylamine is revealed by streptavidin conjugated with peroxidase.
    Figure Legend Snippet: Inhibitory effect of purified MB-MoG-3.7 (grey bars), MB-MoG-2.8 (black bars) and mAb CUB7402 (hatched bars) on mouse tTG activity. Elisa plates coated with gliadin, a tTG substrate, are incubated with 0.2 mM 5-(biotinamido)pentylamine and 0.25 μg of mouse tTG, with increasing amounts of purified miniantibody or mAb. The incorporation of 5-(biotinamido)pentylamine is revealed by streptavidin conjugated with peroxidase.

    Techniques Used: Purification, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation

    ELISA of supernatants of cultured HEK 293T cells transfected with the series of plasmids pMB-SV5 carrying 2.8 scFv gene (A and B) and 3.7 scFv gene (C and D) fused to CH2-CH3 domains genes from human, mouse and rat. Antigens: human tTG, mouse tTG and BSA. Secondary antibodies: A) and C) biotinylated mAb SV5 and streptavidin conjugated with peroxidase; B) and D) goat anti human, mouse and rat IgG or IgA conjugated with peroxidase.
    Figure Legend Snippet: ELISA of supernatants of cultured HEK 293T cells transfected with the series of plasmids pMB-SV5 carrying 2.8 scFv gene (A and B) and 3.7 scFv gene (C and D) fused to CH2-CH3 domains genes from human, mouse and rat. Antigens: human tTG, mouse tTG and BSA. Secondary antibodies: A) and C) biotinylated mAb SV5 and streptavidin conjugated with peroxidase; B) and D) goat anti human, mouse and rat IgG or IgA conjugated with peroxidase.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection

    A, Western blotting of the miniantibody MB-MoG-2.8 with reducing agents (lane 2), treated with glycosidase PNGase F (lane 1), and in not reducing not denaturing conditions (lane 3). B, Immunohistochemistry performed on histological section of mouse muscle tissue with the miniantibodies constructs MB-MoG-2.8 and 3.7. Secondary antibodies: biotinylated mAb SV5 followed by streptavidin conjugated with alkaline phosphatase (western blotting) or horseradish peroxidase (immunohistochemistry).
    Figure Legend Snippet: A, Western blotting of the miniantibody MB-MoG-2.8 with reducing agents (lane 2), treated with glycosidase PNGase F (lane 1), and in not reducing not denaturing conditions (lane 3). B, Immunohistochemistry performed on histological section of mouse muscle tissue with the miniantibodies constructs MB-MoG-2.8 and 3.7. Secondary antibodies: biotinylated mAb SV5 followed by streptavidin conjugated with alkaline phosphatase (western blotting) or horseradish peroxidase (immunohistochemistry).

    Techniques Used: Western Blot, Immunohistochemistry, Construct

    Complement fixation assay with the miniantibodies constructs MB-HuG-2.8 and 3.7, MB-MoG-2.8 and 3.7, MB-RaG-2.8 and 3.7. The binding of C1q to the miniantibodies is revealed with biotinylated anti-C1q and streptavidin conjugated with alkaline phosphatase. Positive and negative control are represented by the murine anti-His D8 (IgG2a) and CUB7402 (IgG1) mAb, both recognizing the coated tTG.
    Figure Legend Snippet: Complement fixation assay with the miniantibodies constructs MB-HuG-2.8 and 3.7, MB-MoG-2.8 and 3.7, MB-RaG-2.8 and 3.7. The binding of C1q to the miniantibodies is revealed with biotinylated anti-C1q and streptavidin conjugated with alkaline phosphatase. Positive and negative control are represented by the murine anti-His D8 (IgG2a) and CUB7402 (IgG1) mAb, both recognizing the coated tTG.

    Techniques Used: Construct, Binding Assay, Negative Control

    ELISA time course of the serum anti-tTG miniantibody average titer in 8 BALB/c mice injected at 0 and 14 days with pMB-MoG-3.7 (panel A) and 2.8 (panel B) DNA. Serum dilution 1:50. Secondary antibodies: biotinylated mAb SV5 and streptavidin conjugated with peroxidase.
    Figure Legend Snippet: ELISA time course of the serum anti-tTG miniantibody average titer in 8 BALB/c mice injected at 0 and 14 days with pMB-MoG-3.7 (panel A) and 2.8 (panel B) DNA. Serum dilution 1:50. Secondary antibodies: biotinylated mAb SV5 and streptavidin conjugated with peroxidase.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    14) Product Images from "Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion"

    Article Title: Altering Expression Levels of Human Immunodeficiency Virus Type 1 gp120-gp41 Affects Efficiency but Not Kinetics of Cell-Cell Fusion

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3522-3533.2002

    Cytosine arabinoside (AraC) treatment reduces cell surface expression of gp120. HeLa cells infected with HXB2 gp160-expressing vaccinia virus were cultured in the presence of various concentrations of cytosine arabinoside and analyzed for surface expression. (A) Western blot detection of biotinylated gp120. Cells infected with HXB2 gp160-expressing vaccinia virus in the presence of the indicated concentrations of AraC were treated with the membrane-impermeant biotinylation reagent sulfo-NHS-LC-biotin, lysed, and immunoprecipitated with NHS (right) or serum from HIV-infected subjects (HIVIG, left). Biotinylated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and detected with horseradish peroxidase-conjugated streptavidin. (B) Cells were prepared as described for panel A but were grown in glass chamber slides and were stained with a phycoerythrin-conjugated anti-gp120 monoclonal antibody.
    Figure Legend Snippet: Cytosine arabinoside (AraC) treatment reduces cell surface expression of gp120. HeLa cells infected with HXB2 gp160-expressing vaccinia virus were cultured in the presence of various concentrations of cytosine arabinoside and analyzed for surface expression. (A) Western blot detection of biotinylated gp120. Cells infected with HXB2 gp160-expressing vaccinia virus in the presence of the indicated concentrations of AraC were treated with the membrane-impermeant biotinylation reagent sulfo-NHS-LC-biotin, lysed, and immunoprecipitated with NHS (right) or serum from HIV-infected subjects (HIVIG, left). Biotinylated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and detected with horseradish peroxidase-conjugated streptavidin. (B) Cells were prepared as described for panel A but were grown in glass chamber slides and were stained with a phycoerythrin-conjugated anti-gp120 monoclonal antibody.

    Techniques Used: Expressing, Infection, Cell Culture, Western Blot, Immunoprecipitation, SDS Page, Staining

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    Article Snippet: .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature. .. Then, 3 amino 9-ethylcarbazol (AEC) solution (Large Volume AEC Substrate System (RTU), Thermo Fisher Scientific, TA-060- HA, Fremont, California, USA) was dropped into the tissues and the tissues were examined by light microscopy.

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody. .. After blocking, the section was incubated overnight with 100-fold diluted primary antibody and rinsed with PBS.

    Article Title: ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
    Article Snippet: .. Next, slides were washed and incubated in streptavidin solution (Thermoscientific cat. no. TS-060-HR). .. This was followed by incubation in AEC and a rinse in H2 O.

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA). .. After the solution of 3-amino-9-ethylcarbazole (AEC) Substrate+AEC Chromogen (AEC Substrate, TA-015 and HAS; AEC Chromogen, TA-002-HAC, Lab Vision Corporation, USA) was added to the tissues and images taken on a light microscope, it was washed with PBS concurrently.

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS. .. After taking the image signal on a light microscope by dripping the solution of 3-amino-9-ethylcarbazole (AEC) Substrate + AEC Chromogen (AEC Substrate, TA-015 and HAS, AEC Chromogen, TA-002-HAC, LabVision Corporation, USA) on tissues, the tissues were washed with PBS synchronously.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Immunohistochemistry Formalin-fixed, paraffin-embedded tissue samples from the surgically resected U87 tumor were cut into 4-mm sections. .. Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody.

    Activity Assay:

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: The endogenous peroxidase activity was blocked with 3% H2 O2 for 15 min at room temperature and later rinsed with distilled water and PBS. .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Immunohistochemistry Sections were dewaxed in xylene and rehydrated through graded alcohols prior to quenching endogenous peroxidase activity with 3% H2 O2 in distilled water for 20 minutes. .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: After washing with PBS for 3×5 minutes, the tissues were incubated in hydrogen peroxide (Hydrogen Peroxide Block, Thermo Fisher Scientific, TA-125-HP, Fremont, California, USA) for 5 minutes to obstruct endogenous peroxidase activity and then washed with PBS for 3×5 minutes. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: After boiling, the tissues were incubated for 5 min with hydrogen peroxide block solution (Hydrogen Peroxide Block, TA-125-HP, Lab Vision Corporation, USA) to prevent endogenous peroxidase activity after washing for 3 × 5 min with PBS (Phosphate-Buffered Saline, P4417, Sigma-Aldrich, USA). .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: After boiling, the tissues that were kept to cool off in room heat for about 20 min were incubated for 5 min with hydrogen peroxide block solution to prevent endogenous peroxidase activity after being washed for 3 × 5 min with PBS (Phosphate Buffered Saline, P4417, Sigma-Aldrich, USA) -125-HP, Lab Vision Corporation, USA). .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS.

    Blocking Assay:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: The sections were washed 3 times for 5 min in PBS and incubated with hydrogen peroxide (catalogue #TA-015-HP, Thermo Fisher Scientific, US) for 20 min. Ultra V block (TA-125-UB, Thermo Fisher Scientific, US) was applied to the sections for 8 min prior to the addition of the primary antibodies, which were left on overnight (osteonectin; SPARC monoclonal antibody, catalogue # 33-5500, 1:100; osteopontin monoclonal antibody, catalogue #MA5-17180, 1:100; and MMP-9 monoclonal antibody, catalogue #MA5-13595, 1:200, all from Thermo Fisher Scientific, US). .. The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: Blocking reagent (Lab Vision, TA-125-UB) was applied to each slide followed by 5 min incubation at room temperature in a humid chamber. .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Non-specific antigen binding was blocked by incubation with UltraVision Protein Block (TA-125- PBQ; Thermo Scientific, Cheshire, UK). .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: Endogenous peroxide was blocked with a 3% H2 O2 solution in methanol for 10 min, and for unmasking antigens, a retrieval solution (Vector Co™) 1% in hot water (98 °C) was added and incubated for 20 min. Ultra V block (TA-125-UB, LabVision Co™) was added to the sample and incubated for 10 min to inhibit nonspecific background staining. .. A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: Ultra V Block (Ultra V Block, TA-125-UB, Fremont, California, USA) was enforced for 5 minutes in order to avoid background staining. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody. .. After blocking, the section was incubated overnight with 100-fold diluted primary antibody and rinsed with PBS.

    Article Title: Enhanced Susceptibility of Ago1/3 Double-Null Mice to Influenza A Virus Infection
    Article Snippet: Endogenous avidin and biotin (catalog number X0590; Dako, Carpinteria, CA) were blocked (10 min each), followed by blocking for 30 min in Superblock (catalog number 37545ZZ; Thermo Scientific Pierce, Rockford, IL). .. Detection of bound secondary antibodies was performed with horseradish peroxidase-labeled streptavidin (catalog number TS-125-HR; Thermo Scientific, Kalamazoo, MI) with 3,3′ diaminobenzidine (DAB; 5 min; catalog number TA-125-HDX; Thermo Shandon) as chromogenic substrate and a light hematoxylin counterstain (catalog number TA-125-MH; Thermo Scientific, Kalamazoo, MI).

    Article Title: ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
    Article Snippet: For cytoplasmic proteins: following antigen retrieval, slides were first blocked with hydrogren peroxidase block (Thermoscientific, TA-060_H202Q), washed and blocked once more with Ultra V block (Thermoscientific, TA-060-PBQ) to reduce non-specific background. .. Next, slides were washed and incubated in streptavidin solution (Thermoscientific cat. no. TS-060-HR).

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: After washing with PBS for 3 × 5 min, a solution of Ultra V Block (TA-125-UB, Lab Vision Corporation, USA) was applied for 5 min to inhibit background dyeing and incubated at room temperature for 60 min in humidified atmosphere with a 1/200 diluted asprosin primer antibody. .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: In order to prevent dye remaining on the tissues that were washed with PBS for 3 × 5 min, Ultra V Block (TA-125-UB, Lab Vision Corporation, USA) solution was applied for 5 min and they were incubated with 1/200 diluted primary antibody (iris Rabbit Polyclonal H-067-17, Phoenix Pharmaceuticals, Inc., California, USA) at room temperature for 60 min in a humid environment. .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS.

    Staining:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: Paragraph title: Immunohistochemical staining ... The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber. .. Both the intensity and distribution of iNOS, IL-6, L-FABP, NGAL staining were scored in 10 different areas for each samples under light microscope at magnification X400 in the kidney slices.

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Collagen deposition was detected using a Masson’s Trichrome stain. .. Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA).

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: Endogenous peroxide was blocked with a 3% H2 O2 solution in methanol for 10 min, and for unmasking antigens, a retrieval solution (Vector Co™) 1% in hot water (98 °C) was added and incubated for 20 min. Ultra V block (TA-125-UB, LabVision Co™) was added to the sample and incubated for 10 min to inhibit nonspecific background staining. .. A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: Ultra V Block (Ultra V Block, TA-125-UB, Fremont, California, USA) was enforced for 5 minutes in order to avoid background staining. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Western Blot:

    Article Title: The Spermatogonial Stem Cell Niche in the Collared Peccary (Tayassu tajacu) 1
    Article Snippet: Paragraph title: Western Blot Analysis ... The strips were washed three times with TBS-0.05% Tween solution, 5 min each, and incubated in streptavidin solution (TS-125-HR; Thermo Scientific) for 15 min, at room temperature.

    Article Title: Spermatogonial Stem Cell Markers and Niche in Equids
    Article Snippet: Paragraph title: Western blotting analysis ... Before exposing the strips to streptavidin solution (Thermo Scientific, TS-125-HR) for 15 minutes at room temperature, they were rinsed three times with TBS 0.05% Triton-X buffer.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Binding Assay:

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: Kidney sections were incubated overnight at 4°C with rabbit polyclonal iNOS antibody (iNOS Ab-1, Rabbit PAb, RB-1605-P, NeoMarkers Fremont, CA) and IL-6 (Abcam, 6672), and incubated for 1 h at room temperature with anti-myeloperoxidase (MPO) antibodies (Myeloperoxidase Ab-1, RB-373-A, NeoMarkers Fremont, CA), Lipocalin 2 antibody (NGAL, neutrophil gelatinase-associated lipocalin) (abcam 41105), polyclonal antibody to rat L-FABP, liver-fatty acid binding protein (HycultBiotect HP8010). .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin. .. Serial sections of canine lymph node were used as positive control.

    Microscopy:

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA). .. Slides were photographed and examined with a Leica DM500 microscope (Leica DFC295).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS. .. Prepared slides were evaluated and photographed on a Leica DM500 microscope (Leica DFC295).

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  • 99
    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
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    99
    Thermo Fisher streptavidin conjugated hrp
    Photoaffinity labeling of various PrP species. <t>Streptavidin-HRP-probed</t> blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.
    Streptavidin Conjugated Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated hrp/product/Thermo Fisher
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated hrp - by Bioz Stars, 2020-03
    99/100 stars
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    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

    Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Early-Responsive Redox-Sensitive Proteins in Arabidopsis

    doi: 10.1021/pr200918f

    Figure Lengend Snippet: Oxidative modification of identified proteins in planta upon H 2 O 2 treatment. Transgenic plants expressing the protein of interest fused with the FLAG tag were vacuum infiltrated with either water (mock) or 5 mM H 2 O 2 . For analysis of AtCIAPIN1, eEF1α, and AtPTP1, free thiols in the total protein were labeled with BIAM during protein extraction. For analysis of AtNAP1;1 and AtPDIL1-1, free thiols in the samples were first alkylated by IAM. Samples were then treated with DTT and newly generated free thiols were labeled by BIAM. After that, FLAG-tagged protein from each sample was affinity purified, separated by SDS-PAGE, and detected by HRP-Conjugated Streptavidin (to determine the amount of BIAM attached to the FLAG-tagged protein) or by the anti-FLAG M2 antibody (to determine the amount of the total recombinant protein).

    Article Snippet: Immunoprecipitated protein was separated by SDS-PAGE and immunoblotted with either anti-FLAG M2-Peroxidase (HRP) antibody (Sigma) or Horseradish Peroxidase-Conjugated Streptavidin (Thermo Scientific).

    Techniques: Modification, Transgenic Assay, Expressing, FLAG-tag, Labeling, Protein Extraction, Generated, Affinity Purification, SDS Page, Recombinant

    Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Journal: Biochemistry

    Article Title: Prion Nucleation Site Unmasked by Transient Interaction with Phospholipid Cofactor

    doi: 10.1021/bi4014825

    Figure Lengend Snippet: Photoaffinity labeling of various PrP species. Streptavidin-HRP-probed blots of samples photoaffinity labeled with PA-PBD peptide. (A) Samples containing PrP Int1 or PrP C were incubated with or without PA-PBD and exposed to UV light for varying time periods, as indicated. (B) Samples containing α -helical PrP or PrP Int1 were incubated with PA-PBD and exposed to UV light for 5 min. (C) Samples of PrP Int1 were incubated with varying concentrations of PA-PBD, as indicated, and exposed to UV light for 0 or 5 min, as indicated. (D) Sample containing 7 μ g of PrP Int1 photoaffinity labeled with PA-PBD (PA-PrP Int1 ) is compared to a standard curve of biotinylated AviTag PrP for reference.

    Article Snippet: The resulting photoaffinity-labeled molecules were run on SDS-PAGE, transferred to PVDF, blocked with a 2.5% solution of bovine serum albumin (Fisher Scientific, Pittsburgh, PA), and incubated with streptavidin-conjugated HRP (ThermoFisher Scientific, Rockford, IL) at a 1:10 000 dilution before being washed with TBST and developed with SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific, Rockford, IL).

    Techniques: Labeling, Incubation

    TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Journal: Nature Communications

    Article Title: Glycogen synthase kinase 3β ubiquitination by TRAF6 regulates TLR3-mediated pro-inflammatory cytokine production

    doi: 10.1038/ncomms7765

    Figure Lengend Snippet: TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production. ( a ) BMDMs were stimulated with 10 μg ml −1 poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. ( b ) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. ( c ) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b . ( d ) Traf6 +/+ and Traf6 −/− 3T3 cells stimulated with 10 μg ml −1 poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. ( e ) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. ( f ) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. ( g ) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t -test (** P

    Article Snippet: Samples were subsequently immunoprecipitated with an anti-GSK3β antibody and separated on SDS–PAGE followed by streptavidin conjugated to HRP (Thermo Fisher Scientific).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Incubation, In Vitro, Real-time Polymerase Chain Reaction, Expressing