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  • 99
    Thermo Fisher streptavidin horseradish peroxidase abc reagent
    Streptavidin Horseradish Peroxidase Abc Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher streptavidin horseradish peroxidase
    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and <t>streptavidin-Alexa555,</t> then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm
    Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase conjugate
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Streptavidin Horseradish Peroxidase Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase complex
    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on <t>streptavidin</t> magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.
    Streptavidin Horseradish Peroxidase Complex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase hrp
    C 2 -ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C 2 -ceramide (C2) (100 μM) or C 2 -dihydroceramide (C 2 H 2 ) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with <t>streptavidin-HRP</t> and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of
    Streptavidin Horseradish Peroxidase Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase shrp
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Streptavidin Horseradish Peroxidase Shrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ultravision streptavidin horseradish peroxidase
    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after <t>streptavidin</t> labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with <t>streptavidin-HRP</t> (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P
    Ultravision Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher streptavidin horseradish peroxidase hrp conjugate
    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Streptavidin Horseradish Peroxidase Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher poly streptavidin horseradish peroxidase
    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Poly Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher streptavidin horseradish peroxidase detection system
    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Streptavidin Horseradish Peroxidase Detection System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher lightshift stabilized streptavidin horseradish peroxidase
    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Lightshift Stabilized Streptavidin Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sg021 streptavidin horseradish peroxidase pierce
    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with <t>streptavidin-HRP</t> (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.
    Sg021 Streptavidin Horseradish Peroxidase Pierce, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp conjugate
    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
    Streptavidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin horseradish peroxidase hrp conjugated antibody
    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with <t>streptavidin-HRP</t> and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith <t>streptavidin-HRP</t> to show that surface biotinylation and protein loading were approximately equal.
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    Quantification of aldehydic lesions and AP sites using ARP and AA3. (A) HeLa DNA was labeled with ARP and vacuum-spotted onto a nylon membrane. Aldehydic lesions in this DNA were quantified using <t>streptavidin-conjugated</t> horseradish peroxidase (HRP) and detected using a chemiluminescent substrate (open bar). In parallel, aldehydic lesions in HeLa DNA were also labeled with AA3 followed by reaction with Cy5 azide and quantification of fluorescence on a nylon membrane (black bar). AA3-labeled aldehydic lesions reacted with Cy5 azide were also quantified in solution using a microplate reader (gray bars). Mean and standard deviation of triplicate samples is shown. (B) HeLa DNA was pre-treated with NaBH 4 to reduce endogenous aldehydic lesions and AP sites were generated by heat and acid treatment for different lengths of time. The AP sites were labeled using ARP or AA3 for labeling and the DNA was spotted onto a nylon membrane and the membranes scanned to quantify AP sites ( Left ). AP sites in the same DNAs were also quantified by reacting them successively with AA3 and Cy5 azide, and measuring fluorescence intensity directly using a microplate reader ( right) . Mean and standard deviation of triplicate samples is shown for each time point.
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    Image Search Results


    Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Journal: BMC Evolutionary Biology

    Article Title: Functional test of PCDHB11, the most human-specific neuronal surface protein

    doi: 10.1186/s12862-016-0652-x

    Figure Lengend Snippet: Expression of protocadherins. GFP was electroporated alone or in combination with protocadherins into K562 cells. a Immunoblotting of cell lysates with anti-GFP shows bands compatible with the expected molecular weights for GFP (27 kDa) and mature PCDHB11-GFP (110 kDa); image representative of three transfections. b Immunoblotting of cell lysates with anti-HA shows bands compatible with the expected molecular weights for mature HA-PCDHB11-GFP (113 kDa) and HA-PCDHGA3 (99 kDa); image representative of two transfections. c Proportion of medium and large clusters was lower in HA-PCDHB11-GFP-transfected than in HA-PCDHGA3-transfected cells, but higher than in control cells ( n = 10-12 images per condition). d Proportion of medium to large cell clusters when normalized by HA-PCDHGA3. For visualization of surface protocadherins, live cells transfected with GFP alone (e) or in combination with HA-PCDHB11-GFP (f) or HA-PCDHGA3 (g) were stained with anti-HA-biotin and streptavidin-Alexa555, then fixed. Blue: DAPI. Green: GFP. Red: surface HA-tagged protocadherins. Scale bar: 10 μm

    Article Snippet: For GFP detection, membranes were incubated with a rabbit polyclonal antibody (Life Technologies, A-11122) diluted 1:2000 in 50 mM Tris, 100 mM NaCl, 0.1 % Tween 20, pH 7.4, with 3 % bovine serum albumine (Sigma, St. Louis, MO) and a horse-radish peroxidase-conjugated anti-rabbit secondary antibody (Invitrogen, Waltham, MA), while for HA revelation, they were incubated with biotinylated 3 F10 rat monoclonal antibody (Roche Life Science, Indianapolis, IN) diluted 1:500 in the same buffer, and streptavidin-horseradish peroxidase (Invitrogen, Waltham, MA).

    Techniques: Expressing, Transfection, Staining

    Generation and characterization of the peptide-Fc fusion proteins. ( a ) Schematic representation of peptide-Fc construction. The arrow indicates the cleavage site of IL-2 signal sequence. A glycine linker (in blue) was added to further increase the flexibility of the peptide moiety. ( b ) Peptide-Fc fusion expression in HEK293T cells. Subsequent to 48 hours of transfection time, culture supernatants (15 µl each) were analyzed by western blots using biotin-conjugated antihuman Fc antibody followed by streptavidin–HRP. Lanes 1 to 4 correspond to cells transfected with plasmid encoding WN-Fc, LTV-Fc, MY-Fc, and Fc control, respectively. Unt, supernatant from untransfeted cells. ( c ) Secreted peptide-Fc fusion proteins were purified with protein G affinity chromatography from culture supernatants and analyzed by SDS-PAGE under reducing conditions and then stained with Coomassie blue. ( d ) A duplicate gel was transferred to nitrocellulose membrane and processed as in b . Lanes 1 to 4 correspond to WN-Fc, LTV-Fc, MY-Fc, and Fc control fusion proteins, respectively. ( e ) Binding of the peptide-Fc fusion proteins to SKBR3. The cells were incubated with either peptide-Fc fusion proteins (blue histograms) or the Fc control (orange histograms) followed by FITC-conjugated antihuman Fc monoclonal antibody and analysis by flow cytometry. All molecules were tested at 10 µg/ml. In the case of competition experiments, the cells were incubated with the peptide (100 µg/ml) first and then with the corresponding peptide-Fc fusion protein (green histograms). Red histograms represent cells stained with only secondary antibody. Data are from one experiment and are representative of at least 10 independent experiments.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells

    doi: 10.1038/mtm.2015.43

    Figure Lengend Snippet: Generation and characterization of the peptide-Fc fusion proteins. ( a ) Schematic representation of peptide-Fc construction. The arrow indicates the cleavage site of IL-2 signal sequence. A glycine linker (in blue) was added to further increase the flexibility of the peptide moiety. ( b ) Peptide-Fc fusion expression in HEK293T cells. Subsequent to 48 hours of transfection time, culture supernatants (15 µl each) were analyzed by western blots using biotin-conjugated antihuman Fc antibody followed by streptavidin–HRP. Lanes 1 to 4 correspond to cells transfected with plasmid encoding WN-Fc, LTV-Fc, MY-Fc, and Fc control, respectively. Unt, supernatant from untransfeted cells. ( c ) Secreted peptide-Fc fusion proteins were purified with protein G affinity chromatography from culture supernatants and analyzed by SDS-PAGE under reducing conditions and then stained with Coomassie blue. ( d ) A duplicate gel was transferred to nitrocellulose membrane and processed as in b . Lanes 1 to 4 correspond to WN-Fc, LTV-Fc, MY-Fc, and Fc control fusion proteins, respectively. ( e ) Binding of the peptide-Fc fusion proteins to SKBR3. The cells were incubated with either peptide-Fc fusion proteins (blue histograms) or the Fc control (orange histograms) followed by FITC-conjugated antihuman Fc monoclonal antibody and analysis by flow cytometry. All molecules were tested at 10 µg/ml. In the case of competition experiments, the cells were incubated with the peptide (100 µg/ml) first and then with the corresponding peptide-Fc fusion protein (green histograms). Red histograms represent cells stained with only secondary antibody. Data are from one experiment and are representative of at least 10 independent experiments.

    Article Snippet: Streptavidin–horseradish peroxidase conjugates were obtained from Thermo Scientific (Rockford, IL).

    Techniques: Sequencing, Expressing, Transfection, Western Blot, Plasmid Preparation, Purification, Affinity Chromatography, SDS Page, Staining, Binding Assay, Incubation, Flow Cytometry, Cytometry

    AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Journal: Analytical Chemistry

    Article Title: Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

    doi: 10.1021/ac500084d

    Figure Lengend Snippet: AMMP and ELISA target-binding assays. (a) Schematic of the AMMP target-binding assay. An HA-tagged ligand binds to target (Bcl-x L ) immobilized on streptavidin magnetic beads. Anti-HA antibody, labeled with fluorescein, binds to the HA tag and to antifluorescein antibody on the sensor surface. (b) AMMP signal in the target-binding assay is a function of dilution factor and ligand affinity. At the same dilution, Pep1 generates higher signal levels than Pep2, whereas ScPep1 and Pep1ΔHA show background levels of signal. (c) AMMP target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay, demonstrating true relative affinities. (d) Schematic of the ELISA target-binding assay. An HA-tagged ligand binds to biotinylated target (Bcl-x L ) and anti-HA antibody on the ELISA plate. Streptavidin-HRP binds to biotin, resulting in an ELISA signal. (e) Target-binding ELISA assay is much less sensitive than the AMMP assay [described in (b)], and the respective samples behave equivalently: Pep1 generates a higher signal level than Pep2 and ScPep1 and Pep1ΔHA generate no signal over the background. (f) ELISA target-binding assay adjusted for peptide concentration as measured using the HA tag competition assay.

    Article Snippet: Plates were washed with wash buffer [1× PBS + 0.1% (v/v) Tween-20] and blocked with 1× PBS + 5% (w/v) BSA for 2 h. In each well, 100 μL of sample and 100 μL of 33 nM biotinylated Bcl-xL were added and incubated for 4 h. Plates were washed, incubated with streptavidin horseradish peroxidase conjugate (Strep-HRP, Thermo Scientific) for 1 h, washed, and incubated with TMB substrate (Thermo Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Magnetic Beads, Labeling, Concentration Assay, Competitive Binding Assay

    C 2 -ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C 2 -ceramide (C2) (100 μM) or C 2 -dihydroceramide (C 2 H 2 ) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with streptavidin-HRP and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Insulin-Stimulated Glucose Transporter GLUT4 Translocation and Akt Kinase Activity by Ceramide

    doi:

    Figure Lengend Snippet: C 2 -ceramide inhibits insulin-stimulated GLUT4 and IRAP translocation. Plasma membrane GLUT4 (A and B) and IRAP (C and D) levels were measured as described in Materials and Methods. In both assays, C 2 -ceramide (C2) (100 μM) or C 2 -dihydroceramide (C 2 H 2 ) (100 μM) was added 2 h prior to initiation of the assay. Insulin (20 nM) was present for the last 10 min. Immunofluorescence detection of GLUT4 on plasma membrane sheets was performed with polyclonal sheep anti-GLUT4 primary antibodies followed by rhodamine-conjugated anti-sheep secondary antibodies. Images were captured with a digital camera (A) and quantitated as described in Materials and Methods (B). Biotinylated IRAP was detected with streptavidin-HRP and visualized with enhanced chemifluorescence (C), and the results were quantitated on a phosphorimager (D). Each asterisk denotes that the difference from the value obtained in the presence of insulin alone was statistically significant at a P value of

    Article Snippet: SDS gels intended for biotin quantitation were transferred to polyvinylidene difluoride membranes (Fisher), blocked in Tris-buffered saline containing 0.2% Tween with 6% bovine serum albumin, treated with 1 μg of streptavidin-horseradish peroxidase (HRP) (Pierce) per ml for 2 h, washed in Tris-buffered saline containing 0.2% Tween, and developed with an enhanced chemifluorescence kit (Amersham) on a STORM 860 scanner.

    Techniques: Translocation Assay, Immunofluorescence

    RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Journal: Molecular and Cellular Biology

    Article Title: Inhibition of ErbB-2 Mitogenic and Transforming Activity by RALT, a Mitogen-Induced Signal Transducer Which Binds to the ErbB-2 Kinase Domain †

    doi:

    Figure Lengend Snippet: RALT binds to SH3 domains. (A) Lysates of 293-RALT transfectants were incubated with the indicated recombinant GST fusion proteins immobilized onto glutathione-agarose beads. Proteins bound to resins were analyzed by immunoblotting with anti-RALT antibodies and the ECL reaction. Lysate in the left lane represents 5% of the input lysate in the binding reaction. (B) GST–GRB-2 fusions expressed from either wt GRB-2 cDNA or cDNA encoding the indicated GRB-2 mutant proteins were purified onto glutathione-agarose beads in similar amounts and used as affinity reagents for the capture of RALT solubilized from 293 transfectants; lysate in the left lane corresponds to 5% input in the binding reaction. Proteins bound to resin were subjected to immunoblot analysis with S1 anti-RALT antibodies. (C) Each of the indicated recombinant proteins (2 μg; purified by affinity chromatography on glutathione-agarose resin) was run on SDS-PAGE gel and transferred to nitrocellulose filters. After the proteins were stained with Ponceau's red to ensure that equal amounts of protein were present in each lane, independent filters were probed with biotin-labeled GST–RALT 1-262 or GST–RALT 263-459; detection was with HRP-conjugated streptavidin followed by the ECL reaction. No specific reactivity was observed with RALT 1-262, and therefore only the filter probed with GST–RALT 263-459 is shown. (D) Lysates from 293 cells transfected with RALT or RALT and GRB-2 expression vectors were analyzed for RALT and GRB-2 expression by immunoblotting (upper two panels). Anti-RALT immunoprecipitates from these lysates were blotted with anti-GRB-2 antibodies along with lysates representing 10% of the input protein in the immunoprecipitation reaction (lower two panels). ECL exposure of the third panel from top was 90 s, while that of the bottom panel was 45 s. WB, Western blotting; Tx, transfection.

    Article Snippet: RALT proteins captured by the filter were detected by horseradish peroxidase (HRP)-conjugated streptavidin (Pierce) (0.1 μg/ml) followed by enhanced chemiluminescence (ECL).

    Techniques: Incubation, Recombinant, Binding Assay, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Staining, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot

    Affinity of STIV-selected ssDNA aptamer interactions by ELISA. STIV was incubated with increasing concentrations of 5′-biotinylated aptamer. After addition of streptavidin-HRP, the amount of STIV-aptamer complex was calculated and graphed as a function of aptamer concentration. The graph was fit to the equation Y = Bmax*X/( K d + X) using SigmaPlot software. Results for each aptamer was presented as the mean ± SD of three independent experiments

    Journal: BMC Veterinary Research

    Article Title: Characterization of DNA aptamers generated against the soft-shelled turtle iridovirus with antiviral effects

    doi: 10.1186/s12917-015-0559-6

    Figure Lengend Snippet: Affinity of STIV-selected ssDNA aptamer interactions by ELISA. STIV was incubated with increasing concentrations of 5′-biotinylated aptamer. After addition of streptavidin-HRP, the amount of STIV-aptamer complex was calculated and graphed as a function of aptamer concentration. The graph was fit to the equation Y = Bmax*X/( K d + X) using SigmaPlot software. Results for each aptamer was presented as the mean ± SD of three independent experiments

    Article Snippet: The bound aptamers were detected using streptavidin-conjugated horseradish peroxidase (HRP) (1:10,000, Pierce).

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Software

    Identification of an anti-ζ-globin chain mAbs pair for detecting ζ-globin chain. An ELISA plate was coated with 10 μg/mL of the anti-ζ-globin chain mAb clones PL2 or PL3 or an isotype-matched control mAb (AM85B-8B) as indicated. A sandwich ELISA for detecting various concentrations of ζ-globin chains was performed using (A) biotin-labeled mAb PL2 (PL2-biotin) and (B) biotin-labeled mAb PL3 (PL3-biotin). HRP-conjugated streptavidin was used to monitor the antigen-antibody reaction.

    Journal: PLoS ONE

    Article Title: Impact of the detection of ζ-globin chains and hemoglobin Bart’s using immunochromatographic strip tests for α0-thalassemia (--SEA) differential diagnosis

    doi: 10.1371/journal.pone.0223996

    Figure Lengend Snippet: Identification of an anti-ζ-globin chain mAbs pair for detecting ζ-globin chain. An ELISA plate was coated with 10 μg/mL of the anti-ζ-globin chain mAb clones PL2 or PL3 or an isotype-matched control mAb (AM85B-8B) as indicated. A sandwich ELISA for detecting various concentrations of ζ-globin chains was performed using (A) biotin-labeled mAb PL2 (PL2-biotin) and (B) biotin-labeled mAb PL3 (PL3-biotin). HRP-conjugated streptavidin was used to monitor the antigen-antibody reaction.

    Article Snippet: Horseradish peroxidase (HRP)-labeled streptavidin and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate were purchased from Invitrogen (Camarillo, CA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, Sandwich ELISA, Labeling

    Specific HA detection after vacuum slot blotting to a positively charged nylon membrane is M -dependent. At the same mass loaded, HA with larger M gives a more intense signal after detection with biotinylated HABP/Streptavidin-HRP/ECL/film. The film was

    Journal: Glycobiology

    Article Title: Molecular mass dependence of hyaluronan detection by sandwich ELISA-like assay and membrane blotting using biotinylated hyaluronan binding protein

    doi: 10.1093/glycob/cwt064

    Figure Lengend Snippet: Specific HA detection after vacuum slot blotting to a positively charged nylon membrane is M -dependent. At the same mass loaded, HA with larger M gives a more intense signal after detection with biotinylated HABP/Streptavidin-HRP/ECL/film. The film was

    Article Snippet: Horseradish peroxidase-conjugated streptavidin (SA-HRP) was from Invitrogen (Carlsbad, CA).

    Techniques:

    CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P

    Journal: Molecular and Cellular Biology

    Article Title: CD9 Regulates Major Histocompatibility Complex Class II Trafficking in Monocyte-Derived Dendritic Cells

    doi: 10.1128/MCB.00202-17

    Figure Lengend Snippet: CD9 regulates MHC-II internalization and recycling in mature MoDCs. (A and B) Immature (A) and LPS-matured (B) WT and CD9 KO MoDCs were incubated with biotinylated MHC-II antibodies for 1 h at 4°C, washed, and incubated for different times at 37°C, and then MHC-II surface expression was detected by flow cytometry after streptavidin labeling in CD11c + cells. Data represent mean fold changes ± SEM of results from three independent experiments performed in triplicate and were analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. (C and D) Measurement of MHC-II internalization (C) and recycling (D) in mature WT and CD9 KO MoDCs by cell biotinylation assay. After incubation with biotin (blue in the schemes) at 4°C, cells were kept at 37°C (internalization), and the remaining surface biotins were removed by MESNA washing. (D) For recycling experiments, cells were further incubated for different times at 37°C (recycling), and surface biotins removed by MESNA washing. Cell lysates were immunoprecipitated with an anti-MHC-II antibody (M5/114), and biotinylated proteins were detected after membrane incubation with streptavidin-HRP (StrepHRP). Membranes were reprobed with MHC-II antibody for loading measurement. The blots shown are from representative experiments. (C) Only a fractional amount from cells incubated at 4°C in the absence of MESNA (0 min) was loaded. The graph shows the biotinylated MHC-II/total MHC-II signal ratio. The graph shows data determined as follows: [1 − (biotinylated MHC-II/total MHC-II signal ratio)]. Data represent mean fold changes ± SEM of results from four (C) and three (D) independent experiments analyzed by two-way ANOVA with Bonferroni's post hoc multiple-comparison test. **, P

    Article Snippet: For internalization and recycling assays, biotinylated proteins were revealed using a Fujifilm LAS-4000 system after membrane incubation with streptavidin-HRP (Invitrogen).

    Techniques: Incubation, Expressing, Flow Cytometry, Cytometry, Labeling, Cell Surface Biotinylation Assay, Immunoprecipitation

    Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.

    Journal: Molecular and Cellular Biology

    Article Title: Dynamic Interplay between Adhesive and Lateral E-Cadherin Dimers

    doi: 10.1128/MCB.22.21.7449-7458.2002

    Figure Lengend Snippet: Metabolic stability of the dimeric and monomeric forms of E-cadherin. (A) Surface proteins of AEcM cells grown in 5-cm-diameter dishes were biotinylated and then chased in regular media. The chase times (in hours) are indicated above the blots. Cells were immunoprecipitated with an excess of anti-myc antibody, and the immunoprecipitates were adjusted to 60 μl with SDS-polyacrylamide gel electrophoresis sample buffer. Five microliters of each immunoprecipitate was loaded. The blots were probed either with streptavidin-HRP (Str-HRP) or anti-myc antibody (Myc). Three different exposure times of the blot developed by Str-HRP are shown. After an exposure time of 10 s [Str-HRP (10)], only the Ec1M protein was visualized, allowing us to estimate the half-life of the monomeric E-cadherin. A longer, 60-s exposure [Str-HRP (60)] visualized coimmunoprecipitated endogenous cadherin (Ec). The blots at the bottom of panel A [Str-HRP (E)] show blots of different exposure times equilibrated on the Ec1M signals, which allowed us to demonstrate that the ratio of Ec1M to endogenous cadherin did not change during the chase periods. Panel B is identical to panel A except that cell lysates before anti-myc immunoprecipitation were subjected to sucrose gradient centrifugation either immediately after the biotinylation (blot 0) or after the 16-h chase (blot 16). The exposure time of blot 0 was shorter than that of blot 16, showing that during the 16-h chase the Ec1M/E-cadherin ratio did not change. Note that immunoprecipitation of Ec1M leads to coimmunoprecipitation of the endogenous E-cadherin (Ec) only in fractions 4 to 6. (C) Surface-biotinylated A-431 cells were dissociated by EGTA and either cocultured for additional 8 h with AEcM cells (lane 1) or cultivated separately and combined after lysis (lane 2). The latter served as a control, showing the absence of interactions between cadherin molecules in solution. The small black bars on the left of the blots indicate the positions of molecular mass markers of 116 and 97.4 kDa.

    Article Snippet: Biotinylated proteins were visualized with streptavidin-horseradish peroxidase (HRP) conjugate (Pierce) in conjunction with ECL (Boehringer Mannheim, Indianapolis, Ind.).

    Techniques: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Gradient Centrifugation, Lysis

    GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Journal: ACS nano

    Article Title: Dynamic Biodistribution of Extracellular Vesicles In Vivo Using a Multimodal Imaging Reporter

    doi: 10.1021/nn404945r

    Figure Lengend Snippet: GlucB and sshBirA label and biotinylate EVs on the surface ( a, b ) Stable HEK293T cells expressing sshBirA with GlucB or Gluc. ( a ) Live-cell imaging of HEK293T cells stably transduced with GlucB-IRES-GFP or Gluc-IRES-GFP vectors, both with sshBirA-IRES-mCherry. Bar, 100 μm. ( b ) Western blot analysis showing enhanced biotinylation of cells stably expressing GlucB with sshBirA, as compared to GlucB alone. No biotinylation was detected in cells expressing Gluc alone or Gluc with sshBirA. Immunoblotting with anti-Gluc antibodies showed Gluc and GlucB at expected sizes (Gluc: 20 kDa; GlucB: 42 kDa). A low level of biotinylated BAP domain (22 kDa) was also detected in GlucB and GlucB + sshBirA samples. Mock transduced HEK293T were used as a negative control. GAPDH was immunoprobed as a loading control. ( c, d ) Transmission electron micrograph (TEM) demonstrating biotinylation and Gluc labeling of EV-GlucB on the membrane. ( c or anti-Gluc antibody followed by gold-conjugated secondary antibody to visualize GlucB labeling of EVs on the membrane, with EV-Gluc showing no Gluc signal but having the CD63 signal. Bar, 100 nm. ( d ) EVs in suspension were immunolabeled with an anti-biotin antibody followed by 10 nm gold-conjugated secondary antibody and biotinylation of EV-GlucB, but not EV-Gluc surface was detected. ( e ) Dot blot detection of biotinylated EVs. EVs isolated from HEK293T cells stably expressing sshBirA with either GlucB (top) or Gluc (bottom) were dot blotted on nitrocellulose membranes in a dose range followed by probing with streptavidin-HRP and chemiluminescence detection. EV-GlucB showed quantity-dependent biotinylated EVs, whereas EV-Gluc control exhibited no biotinylation background signal. ( f ) Nanoparticle tracking analysis (NTA) of EVs. Similar size distribution between EV-Gluc (peaks: 67, 100 and 177 nm) and EV-GlucB (81, 104 and 175 nm) vesicles was detected.

    Article Snippet: Gluc or GlucB labeled EVs in PBS were spotted onto nitrocellulose membranes at 16, 31, 63, 125, 250, 500 and 1,000 ng protein followed by overnight incubation in 5% BSA fraction V (Invitrogen), immunoblotting with streptavidin-HRP conjugate (Pierce) and chemiluminescence detection of biotinylated EVs with SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) on autoradiography films (Denville Scientific, Metuchen, NJ).

    Techniques: Expressing, Live Cell Imaging, Stable Transfection, Transduction, Western Blot, Negative Control, Transmission Assay, Transmission Electron Microscopy, Labeling, Immunolabeling, Dot Blot, Isolation

    Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Journal: Retrovirology

    Article Title: Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses

    doi: 10.1186/1742-4690-5-2

    Figure Lengend Snippet: Effect of glycosylation inhibitors on expression of HA-tagged mCAT-1 in M. dunni cells. (A) Immunoblot analysis of lysates prepared from cells treated for 3 days with the indicated inhibitors: DMM, CST, Sw, DMN (65 μg/ml); 2DG (10 mM); Tu (0.125 ug/ml). (B) Immunoblot of surface biotinylated proteins from DMM-treated and untreated cells. The upper panel was probed with anti-HA; the lower panel shows the same blot stripped and reprobedwith streptavidin-HRP to show that surface biotinylation and protein loading were approximately equal.

    Article Snippet: The membrane was stripped using Pierce Stripping Buffer (catalog no. 21059) and reprobed using HRP-conjugated streptavidin (Pierce; catalog no. 21126).

    Techniques: Expressing

    Quantification of aldehydic lesions and AP sites using ARP and AA3. (A) HeLa DNA was labeled with ARP and vacuum-spotted onto a nylon membrane. Aldehydic lesions in this DNA were quantified using streptavidin-conjugated horseradish peroxidase (HRP) and detected using a chemiluminescent substrate (open bar). In parallel, aldehydic lesions in HeLa DNA were also labeled with AA3 followed by reaction with Cy5 azide and quantification of fluorescence on a nylon membrane (black bar). AA3-labeled aldehydic lesions reacted with Cy5 azide were also quantified in solution using a microplate reader (gray bars). Mean and standard deviation of triplicate samples is shown. (B) HeLa DNA was pre-treated with NaBH 4 to reduce endogenous aldehydic lesions and AP sites were generated by heat and acid treatment for different lengths of time. The AP sites were labeled using ARP or AA3 for labeling and the DNA was spotted onto a nylon membrane and the membranes scanned to quantify AP sites ( Left ). AP sites in the same DNAs were also quantified by reacting them successively with AA3 and Cy5 azide, and measuring fluorescence intensity directly using a microplate reader ( right) . Mean and standard deviation of triplicate samples is shown for each time point.

    Journal: DNA repair

    Article Title: A Versatile New Tool to Quantify Abasic Sites in DNA and Inhibit Base Excision Repair

    doi: 10.1016/j.dnarep.2014.12.006

    Figure Lengend Snippet: Quantification of aldehydic lesions and AP sites using ARP and AA3. (A) HeLa DNA was labeled with ARP and vacuum-spotted onto a nylon membrane. Aldehydic lesions in this DNA were quantified using streptavidin-conjugated horseradish peroxidase (HRP) and detected using a chemiluminescent substrate (open bar). In parallel, aldehydic lesions in HeLa DNA were also labeled with AA3 followed by reaction with Cy5 azide and quantification of fluorescence on a nylon membrane (black bar). AA3-labeled aldehydic lesions reacted with Cy5 azide were also quantified in solution using a microplate reader (gray bars). Mean and standard deviation of triplicate samples is shown. (B) HeLa DNA was pre-treated with NaBH 4 to reduce endogenous aldehydic lesions and AP sites were generated by heat and acid treatment for different lengths of time. The AP sites were labeled using ARP or AA3 for labeling and the DNA was spotted onto a nylon membrane and the membranes scanned to quantify AP sites ( Left ). AP sites in the same DNAs were also quantified by reacting them successively with AA3 and Cy5 azide, and measuring fluorescence intensity directly using a microplate reader ( right) . Mean and standard deviation of triplicate samples is shown for each time point.

    Article Snippet: The membrane was then incubated with Starting Block Blocking Buffer (Fisher) at room temperature for 1Hr, followed by the incubation of streptavidin-conjugated horseradish peroxidase (HRP, Thermo Scientific) in blocking buffer at room temperature for 30 minutes.

    Techniques: Labeling, Fluorescence, Standard Deviation, Generated

    Sensitivity of detection of AP sites using ARP and AA3. (A) Image of the nylon membranes where different amounts of DNA containing a synthetic oligomer with one AP site were labeled using either ARP-HRP (top) or AA3-Cy5 (bottom). (B) Synthetic duplex containing one AP site was labeled with either ARP or AA3. Different dilutions of ARP-labeled DNA was spotted on a membrane and bound with Cy5-streptavidin. AA3-labeled DNA was reacted with Cy5 azide and different dilutions were spotted on a membrane. Cy5 fluorescence was quantified in each case and is plotted against the number of AP sites in each spot. (Inset) Image of the membrane in each case.

    Journal: DNA repair

    Article Title: A Versatile New Tool to Quantify Abasic Sites in DNA and Inhibit Base Excision Repair

    doi: 10.1016/j.dnarep.2014.12.006

    Figure Lengend Snippet: Sensitivity of detection of AP sites using ARP and AA3. (A) Image of the nylon membranes where different amounts of DNA containing a synthetic oligomer with one AP site were labeled using either ARP-HRP (top) or AA3-Cy5 (bottom). (B) Synthetic duplex containing one AP site was labeled with either ARP or AA3. Different dilutions of ARP-labeled DNA was spotted on a membrane and bound with Cy5-streptavidin. AA3-labeled DNA was reacted with Cy5 azide and different dilutions were spotted on a membrane. Cy5 fluorescence was quantified in each case and is plotted against the number of AP sites in each spot. (Inset) Image of the membrane in each case.

    Article Snippet: The membrane was then incubated with Starting Block Blocking Buffer (Fisher) at room temperature for 1Hr, followed by the incubation of streptavidin-conjugated horseradish peroxidase (HRP, Thermo Scientific) in blocking buffer at room temperature for 30 minutes.

    Techniques: Labeling, Fluorescence