hrp-rabbit anti-mouse igg Search Results


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  • 99
    Thermo Fisher anti rabbit
    All morbillivirus V proteins co-precipitate Jak1 and Tyk2. (a) Vero human-SLAM cells (b), (c) HEK 293FT cells were co-transfected with 1 µg of empty vector or plasmids encoding the indicated proteins along with 2 µg pFLAG-Jak1 or pFLAG-Tyk2. Forty-eight hours post-transfection, cells were lysed with NP-40 lysis buffer (pH 7.5) and the lysates were immunoextracted with mouse anti-V5 or mouse anti-FLAG antibodies as described in “ Materials and methods ”. The immunoprecipitates and the total cell lysate (1/10) were analyzed in Western blots for the presence of FLAG-Jak1 or FLAG-Tyk2 and the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse <t>IgG</t> antibody.
    Anti Rabbit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12618 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg whole molecule peroxidase antibody
    HEK293 cells transfected with expression constructs of <t>Flag-wt-</t> SETX ( F-wt ); Flag-L389S- SETX ( F-L389S ); and C14orf178-GFP (C14orf178). In the upper panel, proteins isolated upon GFP immunoprecipitation were immunoblotted with anti-Flag antibody, and a band of ∼75 kDa (arrow) representing Flag-Senataxin is detected, but only when Flag-L389S-SETX is co-transfected with C14orf178-GFP. No interaction is detected for Flag-wt-SETX with C14orf178-GFP. In the lower panel, representing ‘Input’, anti-Flag signal at ∼ 75 kDa (arrow) is detected in lanes 1, 2, 4, and 5, as expected for HEK293 cells transfected with recombinant Flag-tagged senataxin protein, prior to anti-GFP IP. In the upper panel, a non-specific band of variable intensity (arrowhead) is detected by anti-Flag antibody in the GFP immunoprecipitates, and is not Flag-senataxin, as it is present in HEK293 cells not expressing Flag-tagged senataxin, and likely corresponds to <t>IgG</t> heavy chain.
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam rabbit anti mouse igg h l hrp
    Activation of immune response to OVA by plant viruses/SPs. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse <t>HRP</t> conjugate against <t>IgG</t> (ab6728, Abcam, Cambridge, MA, USA). Concentration of OVA on microplate – 10 μg/ml, ● – anti-OVA serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.044. GMT, geometric mean titer; SD, standard deviation.
    Rabbit Anti Mouse Igg H L Hrp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc hrp conjugated anti mouse igg
    Activation of immune response to OVA by plant viruses/SPs. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse <t>HRP</t> conjugate against <t>IgG</t> (ab6728, Abcam, Cambridge, MA, USA). Concentration of OVA on microplate – 10 μg/ml, ● – anti-OVA serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.044. GMT, geometric mean titer; SD, standard deviation.
    Hrp Conjugated Anti Mouse Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 670 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies hrp conjugated rabbit anti mouse igg
    Effects of 18-mer peptides on the interaction of LPS with LBP. (A) LPS-LBP binding was examined by incubating 50 μl of RPMI 1640 containing 0.1, 1, or 10% FBS in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37°C. After incubation, bound LBP was detected by TMB reaction using anti-LBP MAb 6G3 and <t>HRP-conjugated</t> rabbit anti-mouse <t>IgG.</t> (B) LPS-coated microtiter plates were preincubated with either 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37 at 0.025 to 0.2 μg/well in 50 μl of RPMI 1640 for 1 h at 37°C. Thereafter, LPS-LBP binding was determined by incubating 50 μl of RPMI 1640 containing 10% FBS in the microtiter plates as described above. LPS-LBP binding was expressed as a percentage of that obtained by incubation with RPMI 1640 containing 10% FBS in the absence of added peptide. Data are means ± SD from three to eight separate experiments. Values for 18-mer K 15 -V 32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. ∗, P
    Hrp Conjugated Rabbit Anti Mouse Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Santa Cruz Biotechnology mouse anti rabbit igg hrp
    Immunoreactivity of various preparations by indirect ELISA Filled circles: 2C5 antibody, Circles: 2C5-modified doxorubicin-loaded micelles, Filled squares: Isotype-matched <t>IgG,</t> Squares: IgG-modified doxorubicin-loaded micelles . The binding of doxorubicin-loaded PEG-PE micelles harboring 2C5 antibody or isotype antibody to a monolayer of the antigen (nucleohistones) was evaluated by ELISA and detected using an <t>HRP</t> conjugated antibody. Data represent the mean ± SD, n=3.
    Mouse Anti Rabbit Igg Hrp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam goat anti mouse igg h l hrp
    Immunoreactivity of various preparations by indirect ELISA Filled circles: 2C5 antibody, Circles: 2C5-modified doxorubicin-loaded micelles, Filled squares: Isotype-matched <t>IgG,</t> Squares: IgG-modified doxorubicin-loaded micelles . The binding of doxorubicin-loaded PEG-PE micelles harboring 2C5 antibody or isotype antibody to a monolayer of the antigen (nucleohistones) was evaluated by ELISA and detected using an <t>HRP</t> conjugated antibody. Data represent the mean ± SD, n=3.
    Goat Anti Mouse Igg H L Hrp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Boster Bio hrp conjugated anti mouse rabbit igg sabc kit
    Immunoreactivity of various preparations by indirect ELISA Filled circles: 2C5 antibody, Circles: 2C5-modified doxorubicin-loaded micelles, Filled squares: Isotype-matched <t>IgG,</t> Squares: IgG-modified doxorubicin-loaded micelles . The binding of doxorubicin-loaded PEG-PE micelles harboring 2C5 antibody or isotype antibody to a monolayer of the antigen (nucleohistones) was evaluated by ELISA and detected using an <t>HRP</t> conjugated antibody. Data represent the mean ± SD, n=3.
    Hrp Conjugated Anti Mouse Rabbit Igg Sabc Kit, supplied by Boster Bio, used in various techniques. Bioz Stars score: 96/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher hrp conjugated rabbit anti mouse igg
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Hrp Conjugated Rabbit Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Agilent technologies rabbit anti mouse igg hrp
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Rabbit Anti Mouse Igg Hrp, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 91/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti rabbit igg whole molecule peroxidase antibody
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Anti Rabbit Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7032 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega anti rabbit igg h l hrp conjugate
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Anti Rabbit Igg H L Hrp Conjugate, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jackson Immuno peroxidase igg fraction monoclonal mouse anti rabbit igg light chain specific
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Peroxidase Igg Fraction Monoclonal Mouse Anti Rabbit Igg Light Chain Specific, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam hrp conjugated rabbit anti mouse igg
    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by <t>HRP-conjugated</t> anti- mouse <t>IgG</t> antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.
    Hrp Conjugated Rabbit Anti Mouse Igg, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Agilent technologies horseradish peroxidase hrp conjugated rabbit anti mouse igg
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
    Horseradish Peroxidase Hrp Conjugated Rabbit Anti Mouse Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher hrp rabbit anti mouse igg
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
    Hrp Rabbit Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Boster Bio goat anti rabbit igg h l secondary antibody hrp conjugate
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
    Goat Anti Rabbit Igg H L Secondary Antibody Hrp Conjugate, supplied by Boster Bio, used in various techniques. Bioz Stars score: 99/100, based on 1006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc hrp conjugated rabbit anti mouse igg
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
    Hrp Conjugated Rabbit Anti Mouse Igg, supplied by Cell Signaling Technology Inc, 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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    94
    Bio-Rad anti mouse igg hrp
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
    Anti Mouse Igg Hrp, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 295 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Jackson Immuno peroxidase affinipure goat anti rabbit igg
    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using <t>HRP-conjugated</t> rabbit <t>IgG</t> (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).
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    All morbillivirus V proteins co-precipitate Jak1 and Tyk2. (a) Vero human-SLAM cells (b), (c) HEK 293FT cells were co-transfected with 1 µg of empty vector or plasmids encoding the indicated proteins along with 2 µg pFLAG-Jak1 or pFLAG-Tyk2. Forty-eight hours post-transfection, cells were lysed with NP-40 lysis buffer (pH 7.5) and the lysates were immunoextracted with mouse anti-V5 or mouse anti-FLAG antibodies as described in “ Materials and methods ”. The immunoprecipitates and the total cell lysate (1/10) were analyzed in Western blots for the presence of FLAG-Jak1 or FLAG-Tyk2 and the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse IgG antibody.

    Journal: PLoS ONE

    Article Title: Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    doi: 10.1371/journal.pone.0057063

    Figure Lengend Snippet: All morbillivirus V proteins co-precipitate Jak1 and Tyk2. (a) Vero human-SLAM cells (b), (c) HEK 293FT cells were co-transfected with 1 µg of empty vector or plasmids encoding the indicated proteins along with 2 µg pFLAG-Jak1 or pFLAG-Tyk2. Forty-eight hours post-transfection, cells were lysed with NP-40 lysis buffer (pH 7.5) and the lysates were immunoextracted with mouse anti-V5 or mouse anti-FLAG antibodies as described in “ Materials and methods ”. The immunoprecipitates and the total cell lysate (1/10) were analyzed in Western blots for the presence of FLAG-Jak1 or FLAG-Tyk2 and the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse IgG antibody.

    Article Snippet: AlexaFluor 488 or 568-coupled anti-mouse IgG, IgG1, IgG2a or anti-rabbit IgG antibodies were from Invitrogen and the HRP-coupled anti-mouse or anti-rabbit antibodies were from GE Healthcare Life Sciences.

    Techniques: Transfection, Plasmid Preparation, Lysis, Western Blot

    Inhibition of Jak1 and Tyk2 over-expression-induced phosphorylation By morbillivirus V proteins. (a) Vero human-SLAM cells were co-transfected with empty vector or plasmid encoding the indicated protein along with 300 ng pFLAG-Jak1. (b) Vero dog-SLAM cells were co-transfected with empty vector or plasmid encoding the indicated protein along with 400 ng pFLAG-Tyk2. Twenty-four hours post-transfection, the cells were lysed in SDS-PAGE sample buffer containing protease and phosphatase inhibitors as described in “ Materials and methods ” and the levels of phosphorylated Jak1 and phosphorylated Tyk2 were analyzed in Western blots using rabbit anti-Jak1P and rabbit anti-Tyk2P antibodies respectively. The blots were also probed with mouse anti-FLAG and mouse anti-V5 antibodies to analyze the expression levels of FLAG-Jak1 or FLAG-Tyk2 and the expected morbillivirus V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody. PCNA levels served as loading control.

    Journal: PLoS ONE

    Article Title: Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    doi: 10.1371/journal.pone.0057063

    Figure Lengend Snippet: Inhibition of Jak1 and Tyk2 over-expression-induced phosphorylation By morbillivirus V proteins. (a) Vero human-SLAM cells were co-transfected with empty vector or plasmid encoding the indicated protein along with 300 ng pFLAG-Jak1. (b) Vero dog-SLAM cells were co-transfected with empty vector or plasmid encoding the indicated protein along with 400 ng pFLAG-Tyk2. Twenty-four hours post-transfection, the cells were lysed in SDS-PAGE sample buffer containing protease and phosphatase inhibitors as described in “ Materials and methods ” and the levels of phosphorylated Jak1 and phosphorylated Tyk2 were analyzed in Western blots using rabbit anti-Jak1P and rabbit anti-Tyk2P antibodies respectively. The blots were also probed with mouse anti-FLAG and mouse anti-V5 antibodies to analyze the expression levels of FLAG-Jak1 or FLAG-Tyk2 and the expected morbillivirus V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody. PCNA levels served as loading control.

    Article Snippet: AlexaFluor 488 or 568-coupled anti-mouse IgG, IgG1, IgG2a or anti-rabbit IgG antibodies were from Invitrogen and the HRP-coupled anti-mouse or anti-rabbit antibodies were from GE Healthcare Life Sciences.

    Techniques: Inhibition, Over Expression, Transfection, Plasmid Preparation, SDS Page, Western Blot, Expressing

    Morbillivirus V proteins show varied abilities to co-precipitate STAT1 and STAT2. Vero human-SLAM cells were transfected in duplicate wells with 1 µg of empty vector or plasmid driving the expression of the indicated protein. Forty-eight hours post-transfection, one member of each pair was lysed with lysis buffer at pH 7.5 and second with lysis buffer at pH 8.0. The lysates were immunoextracted with mouse anti-V5 antibody as in “ Materials and methods ”. The immunoprecipitates (Co-IP) and a fraction of total cell lysate (1/10th) were analyzed in Western blots for the presence of STAT1 or STAT2 using rabbit anti-STAT1 and rabbit anti-STAT2 antibodies respectively. The blots from total cell lysates were also probed with mouse anti-V5 antibody for analyzing the expression levels of the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody.

    Journal: PLoS ONE

    Article Title: Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    doi: 10.1371/journal.pone.0057063

    Figure Lengend Snippet: Morbillivirus V proteins show varied abilities to co-precipitate STAT1 and STAT2. Vero human-SLAM cells were transfected in duplicate wells with 1 µg of empty vector or plasmid driving the expression of the indicated protein. Forty-eight hours post-transfection, one member of each pair was lysed with lysis buffer at pH 7.5 and second with lysis buffer at pH 8.0. The lysates were immunoextracted with mouse anti-V5 antibody as in “ Materials and methods ”. The immunoprecipitates (Co-IP) and a fraction of total cell lysate (1/10th) were analyzed in Western blots for the presence of STAT1 or STAT2 using rabbit anti-STAT1 and rabbit anti-STAT2 antibodies respectively. The blots from total cell lysates were also probed with mouse anti-V5 antibody for analyzing the expression levels of the expected V proteins. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody.

    Article Snippet: AlexaFluor 488 or 568-coupled anti-mouse IgG, IgG1, IgG2a or anti-rabbit IgG antibodies were from Invitrogen and the HRP-coupled anti-mouse or anti-rabbit antibodies were from GE Healthcare Life Sciences.

    Techniques: Transfection, Plasmid Preparation, Expressing, Lysis, Co-Immunoprecipitation Assay, Western Blot

    RPV-Sa infection blocks IFNα-induced phosphorylation of Jak1/Tyk2. A549 cells were infected with RPV-Sa at a MOI of 5 or left uninfected. Eighteen hours post-infection cells were mock treated or treated with 1000 IU/ml of IFNα for 15 minutes, lysed in SDS-PAGE sample buffer containing protease and phosphatase inhibitors and the levels of phosphorylated Jak1 and Tyk2 were analyzed in Western blots using rabbit anti-Jak1P and rabbit anti-Tyk2P antibodies respectively. The blots were also probed with rabbit anti-Jak1, rabbit anti-Tyk2 and rabbit anti-RPV P protein (MB18) to analyze the total Jak1 and Tyk2 and to confirm RPV infection. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody. PCNA levels served as loading control.

    Journal: PLoS ONE

    Article Title: Morbillivirus V Proteins Exhibit Multiple Mechanisms to Block Type 1 and Type 2 Interferon Signalling Pathways

    doi: 10.1371/journal.pone.0057063

    Figure Lengend Snippet: RPV-Sa infection blocks IFNα-induced phosphorylation of Jak1/Tyk2. A549 cells were infected with RPV-Sa at a MOI of 5 or left uninfected. Eighteen hours post-infection cells were mock treated or treated with 1000 IU/ml of IFNα for 15 minutes, lysed in SDS-PAGE sample buffer containing protease and phosphatase inhibitors and the levels of phosphorylated Jak1 and Tyk2 were analyzed in Western blots using rabbit anti-Jak1P and rabbit anti-Tyk2P antibodies respectively. The blots were also probed with rabbit anti-Jak1, rabbit anti-Tyk2 and rabbit anti-RPV P protein (MB18) to analyze the total Jak1 and Tyk2 and to confirm RPV infection. The primary antibodies were detected with peroxidase-labelled anti-mouse or anti-rabbit IgG antibody. PCNA levels served as loading control.

    Article Snippet: AlexaFluor 488 or 568-coupled anti-mouse IgG, IgG1, IgG2a or anti-rabbit IgG antibodies were from Invitrogen and the HRP-coupled anti-mouse or anti-rabbit antibodies were from GE Healthcare Life Sciences.

    Techniques: Infection, SDS Page, Western Blot

    HEK293 cells transfected with expression constructs of Flag-wt- SETX ( F-wt ); Flag-L389S- SETX ( F-L389S ); and C14orf178-GFP (C14orf178). In the upper panel, proteins isolated upon GFP immunoprecipitation were immunoblotted with anti-Flag antibody, and a band of ∼75 kDa (arrow) representing Flag-Senataxin is detected, but only when Flag-L389S-SETX is co-transfected with C14orf178-GFP. No interaction is detected for Flag-wt-SETX with C14orf178-GFP. In the lower panel, representing ‘Input’, anti-Flag signal at ∼ 75 kDa (arrow) is detected in lanes 1, 2, 4, and 5, as expected for HEK293 cells transfected with recombinant Flag-tagged senataxin protein, prior to anti-GFP IP. In the upper panel, a non-specific band of variable intensity (arrowhead) is detected by anti-Flag antibody in the GFP immunoprecipitates, and is not Flag-senataxin, as it is present in HEK293 cells not expressing Flag-tagged senataxin, and likely corresponds to IgG heavy chain.

    Journal: PLoS ONE

    Article Title: Protein Interaction Analysis of Senataxin and the ALS4 L389S Mutant Yields Insights into Senataxin Post-Translational Modification and Uncovers Mutant-Specific Binding with a Brain Cytoplasmic RNA-Encoded Peptide

    doi: 10.1371/journal.pone.0078837

    Figure Lengend Snippet: HEK293 cells transfected with expression constructs of Flag-wt- SETX ( F-wt ); Flag-L389S- SETX ( F-L389S ); and C14orf178-GFP (C14orf178). In the upper panel, proteins isolated upon GFP immunoprecipitation were immunoblotted with anti-Flag antibody, and a band of ∼75 kDa (arrow) representing Flag-Senataxin is detected, but only when Flag-L389S-SETX is co-transfected with C14orf178-GFP. No interaction is detected for Flag-wt-SETX with C14orf178-GFP. In the lower panel, representing ‘Input’, anti-Flag signal at ∼ 75 kDa (arrow) is detected in lanes 1, 2, 4, and 5, as expected for HEK293 cells transfected with recombinant Flag-tagged senataxin protein, prior to anti-GFP IP. In the upper panel, a non-specific band of variable intensity (arrowhead) is detected by anti-Flag antibody in the GFP immunoprecipitates, and is not Flag-senataxin, as it is present in HEK293 cells not expressing Flag-tagged senataxin, and likely corresponds to IgG heavy chain.

    Article Snippet: To detect FLAG epitope, anti-FLAG M2 antibody (Sigma: F1804) was used (1∶5,000) in conjunction with anti-mouse HRP secondary antibodies (Sigma: A9044) (1∶10,000).

    Techniques: Transfection, Expressing, Construct, Isolation, Immunoprecipitation, Recombinant

    Activation of immune response to OVA by plant viruses/SPs. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of OVA on microplate – 10 μg/ml, ● – anti-OVA serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.044. GMT, geometric mean titer; SD, standard deviation.

    Journal: Scientific Reports

    Article Title: Plant virus particles with various shapes as potential adjuvants

    doi: 10.1038/s41598-020-67023-4

    Figure Lengend Snippet: Activation of immune response to OVA by plant viruses/SPs. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of OVA on microplate – 10 μg/ml, ● – anti-OVA serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.044. GMT, geometric mean titer; SD, standard deviation.

    Article Snippet: Anti-mouse HRP conjugates (Abcam, Cambridge, MA, USA) against IgG (ab6728), IgG1(ab97240), IgG2a (ab97245) and IgG2b (ab97250) were used in a dilution of 1:10000 and anti-mouse HRP conjugate against IgG3 (ab97260) was used in a dilution of 1:5000.

    Techniques: Activation Assay, Mouse Assay, Indirect ELISA, Concentration Assay, Standard Deviation

    Analysis of total IgG titers to potential adjuvants after immunisation with OVA-plant virus particles compositions. ( a) total IgG titers to CaMV; ( b) total IgG titers to PVX; c , total IgG titers to SPs; ( d) total IgG titers to TMV. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of TMV, SPs, PVX on microplate – 10 μg/ml of CaMV – 1 μg/ml, ● – serum log 10 titer from each mouse, – mean. A Wilcoxon-Mann-Whitney two-tailed test was used to compare the statistical differences among the groups. GMT, geometric mean titer; SD, standard deviation.

    Journal: Scientific Reports

    Article Title: Plant virus particles with various shapes as potential adjuvants

    doi: 10.1038/s41598-020-67023-4

    Figure Lengend Snippet: Analysis of total IgG titers to potential adjuvants after immunisation with OVA-plant virus particles compositions. ( a) total IgG titers to CaMV; ( b) total IgG titers to PVX; c , total IgG titers to SPs; ( d) total IgG titers to TMV. Groups of mice were immunised s.c. four times (days 0, 15, 29, 43). Blood was collected after the fourth immunisation on the 56 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of TMV, SPs, PVX on microplate – 10 μg/ml of CaMV – 1 μg/ml, ● – serum log 10 titer from each mouse, – mean. A Wilcoxon-Mann-Whitney two-tailed test was used to compare the statistical differences among the groups. GMT, geometric mean titer; SD, standard deviation.

    Article Snippet: Anti-mouse HRP conjugates (Abcam, Cambridge, MA, USA) against IgG (ab6728), IgG1(ab97240), IgG2a (ab97245) and IgG2b (ab97250) were used in a dilution of 1:10000 and anti-mouse HRP conjugate against IgG3 (ab97260) was used in a dilution of 1:5000.

    Techniques: Mouse Assay, Indirect ELISA, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Standard Deviation

    Analysis of IgG titers to HEL. Groups of mice were immunised s.c. three times (days 0, 15, 29). Blood was collected after the third immunisation on the 46 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of HEL on microplate – 150 μg/ml, ● – anti-HEL serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.037. GMT, geometric mean titer, SD, standard deviation.

    Journal: Scientific Reports

    Article Title: Plant virus particles with various shapes as potential adjuvants

    doi: 10.1038/s41598-020-67023-4

    Figure Lengend Snippet: Analysis of IgG titers to HEL. Groups of mice were immunised s.c. three times (days 0, 15, 29). Blood was collected after the third immunisation on the 46 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of HEL on microplate – 150 μg/ml, ● – anti-HEL serum log 10 titer from each mouse, – mean. P values were calculated using a post hoc Dunn’s multiple-comparison test, which was conducted after a Kruskal-Wallis test. Kruskal-Wallis test P value: 0.037. GMT, geometric mean titer, SD, standard deviation.

    Article Snippet: Anti-mouse HRP conjugates (Abcam, Cambridge, MA, USA) against IgG (ab6728), IgG1(ab97240), IgG2a (ab97245) and IgG2b (ab97250) were used in a dilution of 1:10000 and anti-mouse HRP conjugate against IgG3 (ab97260) was used in a dilution of 1:5000.

    Techniques: Mouse Assay, Indirect ELISA, Concentration Assay, Standard Deviation

    Analysis of total IgG titers to plant viruses after immunisation with HEL-plant virus compositions. ( a ) Total IgG titers to BMMV; ( b) total IgG titers to PVX; ( c ) total IgG titers to CaMV. Groups of mice were immunised s.c. three times (days 0, 15, 29). Blood was collected after the third immunisation on the 46 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of BMMV and PVX on microplate – 10 μg/ml of CaMV – 1 μg/ml, ● – serum log 10 titer from each mouse, – mean. A Wilcoxon-Mann-Whitney two-tailed test was used to compare the statistical difference between groups. GMT, geometric mean titer, SD, standard deviation.

    Journal: Scientific Reports

    Article Title: Plant virus particles with various shapes as potential adjuvants

    doi: 10.1038/s41598-020-67023-4

    Figure Lengend Snippet: Analysis of total IgG titers to plant viruses after immunisation with HEL-plant virus compositions. ( a ) Total IgG titers to BMMV; ( b) total IgG titers to PVX; ( c ) total IgG titers to CaMV. Groups of mice were immunised s.c. three times (days 0, 15, 29). Blood was collected after the third immunisation on the 46 th day of the experiment. Sera titers were evaluated using an indirect ELISA with anti-mouse HRP conjugate against IgG (ab6728, Abcam, Cambridge, MA, USA). Concentration of BMMV and PVX on microplate – 10 μg/ml of CaMV – 1 μg/ml, ● – serum log 10 titer from each mouse, – mean. A Wilcoxon-Mann-Whitney two-tailed test was used to compare the statistical difference between groups. GMT, geometric mean titer, SD, standard deviation.

    Article Snippet: Anti-mouse HRP conjugates (Abcam, Cambridge, MA, USA) against IgG (ab6728), IgG1(ab97240), IgG2a (ab97245) and IgG2b (ab97250) were used in a dilution of 1:10000 and anti-mouse HRP conjugate against IgG3 (ab97260) was used in a dilution of 1:5000.

    Techniques: Mouse Assay, Indirect ELISA, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Standard Deviation

    Effects of 18-mer peptides on the interaction of LPS with LBP. (A) LPS-LBP binding was examined by incubating 50 μl of RPMI 1640 containing 0.1, 1, or 10% FBS in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37°C. After incubation, bound LBP was detected by TMB reaction using anti-LBP MAb 6G3 and HRP-conjugated rabbit anti-mouse IgG. (B) LPS-coated microtiter plates were preincubated with either 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37 at 0.025 to 0.2 μg/well in 50 μl of RPMI 1640 for 1 h at 37°C. Thereafter, LPS-LBP binding was determined by incubating 50 μl of RPMI 1640 containing 10% FBS in the microtiter plates as described above. LPS-LBP binding was expressed as a percentage of that obtained by incubation with RPMI 1640 containing 10% FBS in the absence of added peptide. Data are means ± SD from three to eight separate experiments. Values for 18-mer K 15 -V 32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. ∗, P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

    doi: 10.1128/CDLI.9.5.972-982.2002

    Figure Lengend Snippet: Effects of 18-mer peptides on the interaction of LPS with LBP. (A) LPS-LBP binding was examined by incubating 50 μl of RPMI 1640 containing 0.1, 1, or 10% FBS in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37°C. After incubation, bound LBP was detected by TMB reaction using anti-LBP MAb 6G3 and HRP-conjugated rabbit anti-mouse IgG. (B) LPS-coated microtiter plates were preincubated with either 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37 at 0.025 to 0.2 μg/well in 50 μl of RPMI 1640 for 1 h at 37°C. Thereafter, LPS-LBP binding was determined by incubating 50 μl of RPMI 1640 containing 10% FBS in the microtiter plates as described above. LPS-LBP binding was expressed as a percentage of that obtained by incubation with RPMI 1640 containing 10% FBS in the absence of added peptide. Data are means ± SD from three to eight separate experiments. Values for 18-mer K 15 -V 32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. ∗, P

    Article Snippet: The MAb solution was rinsed out, and 50 μl of HRP-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark; diluted 1,000-fold in PBS containing 0.1% BSA)/well was incubated for 1 h at room temperature.

    Techniques: Binding Assay, Incubation

    Evaluation of LPS-binding activities of 18-mer peptides. (A) LPS-binding activities of the peptides were investigated by incubating 0.02 to 0.5 μg of 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37 in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37°C in 50 μl of RPMI 1640. Bound peptides were detected by TMB reaction by using a rabbit anti-CAP18 Ab and HRP-conjugated goat anti-rabbit IgG. (B) The peptide (0.1 μg of either 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37) was incubated in LPS-coated 96-well microtiter plates for 1 h at 37°C in the absence or presence of added LPS (0.05 to 2.5 μg/well) in 50 μl of RPMI 1640, and bound peptides were detected as described above. Binding of peptides to LPS-coated plates was expressed as a percentage of that observed when 0.5 μg (for panel A) or 0.1 μg (for panel B) of each peptide was incubated in the absence of added LPS. Data are means ± SD from three to five separate experiments. Values for 18-mer K 15 -V 32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. ∗, P

    Journal: Clinical and Diagnostic Laboratory Immunology

    Article Title: Augmentation of the Lipopolysaccharide-Neutralizing Activities of Human Cathelicidin CAP18/LL-37-Derived Antimicrobial Peptides by Replacement with Hydrophobic and Cationic Amino Acid Residues

    doi: 10.1128/CDLI.9.5.972-982.2002

    Figure Lengend Snippet: Evaluation of LPS-binding activities of 18-mer peptides. (A) LPS-binding activities of the peptides were investigated by incubating 0.02 to 0.5 μg of 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37 in LPS-coated 96-well microtiter plates (100 ng of LPS/well) for 1 h at 37°C in 50 μl of RPMI 1640. Bound peptides were detected by TMB reaction by using a rabbit anti-CAP18 Ab and HRP-conjugated goat anti-rabbit IgG. (B) The peptide (0.1 μg of either 18-mer K 15 -V 32 , 18-mer LL, 18-mer LLKKK, or LL-37) was incubated in LPS-coated 96-well microtiter plates for 1 h at 37°C in the absence or presence of added LPS (0.05 to 2.5 μg/well) in 50 μl of RPMI 1640, and bound peptides were detected as described above. Binding of peptides to LPS-coated plates was expressed as a percentage of that observed when 0.5 μg (for panel A) or 0.1 μg (for panel B) of each peptide was incubated in the absence of added LPS. Data are means ± SD from three to five separate experiments. Values for 18-mer K 15 -V 32 are compared with those for 18-mer LL, 18-mer LLKKK, or LL-37. ∗, P

    Article Snippet: The MAb solution was rinsed out, and 50 μl of HRP-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark; diluted 1,000-fold in PBS containing 0.1% BSA)/well was incubated for 1 h at room temperature.

    Techniques: Binding Assay, Incubation

    Immunoreactivity of various preparations by indirect ELISA Filled circles: 2C5 antibody, Circles: 2C5-modified doxorubicin-loaded micelles, Filled squares: Isotype-matched IgG, Squares: IgG-modified doxorubicin-loaded micelles . The binding of doxorubicin-loaded PEG-PE micelles harboring 2C5 antibody or isotype antibody to a monolayer of the antigen (nucleohistones) was evaluated by ELISA and detected using an HRP conjugated antibody. Data represent the mean ± SD, n=3.

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model

    doi: 10.1016/j.jconrel.2012.09.003

    Figure Lengend Snippet: Immunoreactivity of various preparations by indirect ELISA Filled circles: 2C5 antibody, Circles: 2C5-modified doxorubicin-loaded micelles, Filled squares: Isotype-matched IgG, Squares: IgG-modified doxorubicin-loaded micelles . The binding of doxorubicin-loaded PEG-PE micelles harboring 2C5 antibody or isotype antibody to a monolayer of the antigen (nucleohistones) was evaluated by ELISA and detected using an HRP conjugated antibody. Data represent the mean ± SD, n=3.

    Article Snippet: Mouse monoclonal anti Bcl-2 antibody and rabbit anti-mouse IgG-HRP were from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Indirect ELISA, Modification, Binding Assay, Enzyme-linked Immunosorbent Assay

    The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by HRP-conjugated anti- mouse IgG antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.

    Journal: PLoS Pathogens

    Article Title: Molecular anatomy of the receptor binding module of a bacteriophage long tail fiber

    doi: 10.1371/journal.ppat.1008193

    Figure Lengend Snippet: The LTF needle binds to both LPS and OmpC receptors. (A) SDS-PAGE profiles of purified LTF needle and OmpC trimers. “+” and “-” represent boiling in the presence of SDS. The boiled gp37 sample (lane 2) showed the major 27 kDa monomer band and a faint band of residual trimer present in the boiled sample. The unboiled gp37 sample (lane 3) showed two trimer bands, which probably represent two trimer conformations, intact trimer and partially unfolded trimer. (B) Diazirine-labelled gp37 was incubated with purified LPS from E . coli B for 1 h at 37ºC followed by UV irradiation for 15 min and Superose 6 10/300 GL size-exclusion chromatography (red). Same amounts of diazirine-labelled gp37 (blue) and LPS (green) were loaded on the same column as controls. (C) SDS-PAGE profile of the peak fractions from panel B. The gel was developed by silver staining. Lanes 2 to 13 (elution volumes corresponding to 8 to 16 ml) show fractions of gp37-LPS complex peak. Lane 1 shows LPS control peak (elution volume, 9 ml) and lane 14 shows the gp37 control peak (elution volume, 16 ml). Note the presence of a small amount of cross-linked dimer of gp37 of trimers in lane 14. (D) ELISA assay to capture gp37-OmpC complex. The wells were coated with gp37 and OmpC was added at various molar ratios. The unbound OmpC was removed by repeated washing and the amount of bound OmpC was determined by adding mouse anti-OmpC antibody followed by HRP-conjugated anti- mouse IgG antibody. Various controls (coating buffer + OmpC; BSA + OmpC; or gp37 + OmpC + BSA) were included to validate the specific interaction between gp37 and OmpC.

    Article Snippet: After four washes with PBS-T, the wells were incubated with 1:5,000 diluted HRP-conjugated rabbit anti-mouse IgG (Invitrogen, Camarillo, CA) at 37°C for 1 hr.

    Techniques: SDS Page, Purification, Incubation, Irradiation, Size-exclusion Chromatography, Silver Staining, Enzyme-linked Immunosorbent Assay

    Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using HRP-conjugated rabbit IgG (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).

    Journal: Infection and Immunity

    Article Title: Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

    doi: 10.1128/IAI.03122-14

    Figure Lengend Snippet: Staphylococcus aureus LAC* releases SpA. S. aureus was grown to an OD 600 of 1.2. Cell wall extracts (CW) were diluted 1:5 prior to loading on the gel, and supernatant samples (SN) were not diluted. Protein A was detected using HRP-conjugated rabbit IgG (A), SdrE was detected using anti-SdrE IgG (B), and V8 was detected using anti-V8 serum (C). S. aureus LAC* sbi was grown to an OD 600 of 0.3 or 1.2 as indicated (D). Cell wall extracts were diluted 1:20, and supernatant samples were not diluted. Size markers are indicated (kDa).

    Article Snippet: Blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:2,000 or 1:500; Dako) to detect SpA.

    Techniques:

    LytM contributes to release of protein A by LAC*. (A and B) Culture supernatants from LAC* sbi and LAC* sbi lytM grown to the OD 600 indicated were probed with HRP-labeled rabbit IgG in Western immunoblotting. Supernatants harvested at an OD 600 of 0.3 were concentrated 8-fold before being loaded on a gel, and supernatants harvested at an OD 600 of 1.2 were concentrated 2-fold. Size markers are indicated (kDa). (C) Protein A on the surface of LAC* sbi and LAC* sbi lytM was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured by flow cytometry. Values are plotted as a percentage of the mean fluorescence intensity measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. (D) Protein was captured from culture supernatants using chicken anti-SpA polyclonal IgY and detected using biotin-conjugated mouse monoclonal anti-SpA IgG followed by streptavidin-HRP in an ELISA. Values are expressed as a percentage of total released SpA measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean percent release from four independent experiments. Error bars represent the standard errors of the means. **, P = 0.006; ***, P = 0.0003; ns, not significant ( P > 0.05).

    Journal: Infection and Immunity

    Article Title: Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

    doi: 10.1128/IAI.03122-14

    Figure Lengend Snippet: LytM contributes to release of protein A by LAC*. (A and B) Culture supernatants from LAC* sbi and LAC* sbi lytM grown to the OD 600 indicated were probed with HRP-labeled rabbit IgG in Western immunoblotting. Supernatants harvested at an OD 600 of 0.3 were concentrated 8-fold before being loaded on a gel, and supernatants harvested at an OD 600 of 1.2 were concentrated 2-fold. Size markers are indicated (kDa). (C) Protein A on the surface of LAC* sbi and LAC* sbi lytM was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured by flow cytometry. Values are plotted as a percentage of the mean fluorescence intensity measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. (D) Protein was captured from culture supernatants using chicken anti-SpA polyclonal IgY and detected using biotin-conjugated mouse monoclonal anti-SpA IgG followed by streptavidin-HRP in an ELISA. Values are expressed as a percentage of total released SpA measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean percent release from four independent experiments. Error bars represent the standard errors of the means. **, P = 0.006; ***, P = 0.0003; ns, not significant ( P > 0.05).

    Article Snippet: Blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:2,000 or 1:500; Dako) to detect SpA.

    Techniques: Labeling, Western Blot, Fluorescence, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Release of protein A into S. aureus culture supernatants can be inhibited by altering the sorting signal. (A) Protein A on the surface of LAC* spa sbi (pSpA) (black bars) and LAC* spa sbi (pSpAΩSdrESS) (white bars) was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured using flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* spa sbi (pSpA) grown in broth supplemented with ATc (312.5 ng/ml). Bars represent the mean of three independent experiments. Error bars represent the standard errors of the means. ns, not significant ( P > 0.05). (B) LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) were grown in broth supplemented with ATc (312.5 ng/ml), and culture supernatants were probed with HRP-conjugated rabbit IgG. Size markers are indicated (kDa). (C) Quantification of SpA in culture supernatants of LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) by ELISA. Bacteria were grown in broth supplemented with ATc (312.5 ng/ml), and SpA was captured from culture supernatants using chicken anti-SpA polyclonal IgY. Bound SpA was detected using biotin-labeled mouse monoclonal anti-SpA IgG and HRP-conjugated streptavidin in an ELISA. The absorbance at 450 nm was measured, and readings from wells incubated with culture supernatants from LAC* spa sbi (pRMC2) were subtracted from the mean readings for LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) to account for background. Values for LAC* spa sbi (pSpA ΩSdrESS) are expressed as a percentage of the values measured for LAC* spa sbi (pSpA). Bars represent the mean of three independent experiments, and error bars represent the standard errors of the means. ***, P

    Journal: Infection and Immunity

    Article Title: Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

    doi: 10.1128/IAI.03122-14

    Figure Lengend Snippet: Release of protein A into S. aureus culture supernatants can be inhibited by altering the sorting signal. (A) Protein A on the surface of LAC* spa sbi (pSpA) (black bars) and LAC* spa sbi (pSpAΩSdrESS) (white bars) was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured using flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* spa sbi (pSpA) grown in broth supplemented with ATc (312.5 ng/ml). Bars represent the mean of three independent experiments. Error bars represent the standard errors of the means. ns, not significant ( P > 0.05). (B) LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) were grown in broth supplemented with ATc (312.5 ng/ml), and culture supernatants were probed with HRP-conjugated rabbit IgG. Size markers are indicated (kDa). (C) Quantification of SpA in culture supernatants of LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) by ELISA. Bacteria were grown in broth supplemented with ATc (312.5 ng/ml), and SpA was captured from culture supernatants using chicken anti-SpA polyclonal IgY. Bound SpA was detected using biotin-labeled mouse monoclonal anti-SpA IgG and HRP-conjugated streptavidin in an ELISA. The absorbance at 450 nm was measured, and readings from wells incubated with culture supernatants from LAC* spa sbi (pRMC2) were subtracted from the mean readings for LAC* spa sbi (pSpA) and LAC* spa sbi (pSpAΩSdrESS) to account for background. Values for LAC* spa sbi (pSpA ΩSdrESS) are expressed as a percentage of the values measured for LAC* spa sbi (pSpA). Bars represent the mean of three independent experiments, and error bars represent the standard errors of the means. ***, P

    Article Snippet: Blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:2,000 or 1:500; Dako) to detect SpA.

    Techniques: Labeling, Fluorescence, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Incubation

    Replacing the SpA sorting signal with the sorting signal from SdrE reduces release of SpA by LAC*. (A) Protein A on the surface of LAC* sbi , LAC* sbi [SpAΩSdrESS], and LAC* sbi lytM [SpAΩSdrESS] was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured using flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. (B) Protein A was captured from culture supernatants using chicken anti-SpA polyclonal IgY and detected using biotin-conjugated mouse monoclonal anti-SpA IgG followed by streptavidin-HRP in an ELISA. The absorbance at 450 nm was measured, and the mean reading from wells incubated with culture supernatants from LAC* spa sbi were subtracted from the readings for all other wells to account for background. Values are expressed as a percentage of total released SpA measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean of four independent experiments. Error bars represent the standard errors of the means. **, P = 0.007; ***, P

    Journal: Infection and Immunity

    Article Title: Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

    doi: 10.1128/IAI.03122-14

    Figure Lengend Snippet: Replacing the SpA sorting signal with the sorting signal from SdrE reduces release of SpA by LAC*. (A) Protein A on the surface of LAC* sbi , LAC* sbi [SpAΩSdrESS], and LAC* sbi lytM [SpAΩSdrESS] was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured using flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. (B) Protein A was captured from culture supernatants using chicken anti-SpA polyclonal IgY and detected using biotin-conjugated mouse monoclonal anti-SpA IgG followed by streptavidin-HRP in an ELISA. The absorbance at 450 nm was measured, and the mean reading from wells incubated with culture supernatants from LAC* spa sbi were subtracted from the readings for all other wells to account for background. Values are expressed as a percentage of total released SpA measured for LAC* sbi grown to an OD 600 of 0.3. Bars represent the mean of four independent experiments. Error bars represent the standard errors of the means. **, P = 0.007; ***, P

    Article Snippet: Blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:2,000 or 1:500; Dako) to detect SpA.

    Techniques: Labeling, Fluorescence, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Incubation

    Release of D3D4 reporter protein into S. aureus culture supernatants can be inhibited by altering the sorting signal. (A) Amino acid sequences of SpA and SdrE sorting signals. Amino acid coordinates are indicated. Cell wall extracts (B) and culture supernatants (C) from LAC* spa sbi (pD3D4-SpASS) and LAC* spa sbi (pD3D4-SdrESS) were probed with rabbit anti-D3D4 IgG. Bound antibody was detected using HRP-conjugated goat anti-rabbit IgG. Size markers are indicated (kDa).

    Journal: Infection and Immunity

    Article Title: Protein A Is Released into the Staphylococcus aureus Culture Supernatant with an Unprocessed Sorting Signal

    doi: 10.1128/IAI.03122-14

    Figure Lengend Snippet: Release of D3D4 reporter protein into S. aureus culture supernatants can be inhibited by altering the sorting signal. (A) Amino acid sequences of SpA and SdrE sorting signals. Amino acid coordinates are indicated. Cell wall extracts (B) and culture supernatants (C) from LAC* spa sbi (pD3D4-SpASS) and LAC* spa sbi (pD3D4-SdrESS) were probed with rabbit anti-D3D4 IgG. Bound antibody was detected using HRP-conjugated goat anti-rabbit IgG. Size markers are indicated (kDa).

    Article Snippet: Blots were probed with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:2,000 or 1:500; Dako) to detect SpA.

    Techniques: