hrp conjugate Thermo Fisher Search Results


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  • 90
    Thermo Fisher horse radish peroxidase conjugated monoclonal antibody thermo fisher scientific no a10654
    The <t>IgG4</t> responses to O. volvulus -specific antigen (OvAg) in correlation with the age (in years) of the study participants.
    Horse Radish Peroxidase Conjugated Monoclonal Antibody Thermo Fisher Scientific No A10654, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher horse radish peroxidase conjugated monoclonal antibody thermo fisher scientific no
    The <t>IgG4</t> responses to O. volvulus -specific antigen (OvAg) in correlation with the age (in years) of the study participants.
    Horse Radish Peroxidase Conjugated Monoclonal Antibody Thermo Fisher Scientific No, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugate
    The <t>IgG4</t> responses to O. volvulus -specific antigen (OvAg) in correlation with the age (in years) of the study participants.
    Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hisprobe horseradish peroxidase
    3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a <t>HisProbe-HRP</t> Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.
    Hisprobe Horseradish Peroxidase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher probe hrp conjugate
    3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a <t>HisProbe-HRP</t> Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.
    Probe Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher αv5 hrp conjugate
    3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a <t>HisProbe-HRP</t> Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.
    αv5 Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher neutravidin hrp conjugate
    Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with <t>Neutravidin-HRP</t> conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.
    Neutravidin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher antigoat hrp conjugate
    Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with <t>Neutravidin-HRP</t> conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.
    Antigoat Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp enzyme conjugates
    Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with <t>Neutravidin-HRP</t> conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.
    Hrp Enzyme Conjugates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptaviden hrp conjugate
    Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with <t>Neutravidin-HRP</t> conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.
    Streptaviden Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher igg2α hrp conjugates
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Igg2α Hrp Conjugates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 2 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: 90/100, based on 626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated
    FBXO27 is localized to membranes via N-myristoylation. ( A ) FBXO27 binds to glycoproteins modified with high-mannose N -glycans and TFRC. 293T and HeLa cells were transfected with plasmids encoding either only the <t>FLAG</t> tag, or FLAG-tagged FBXO2 (O2), FBXO6 (O6), FBXO6Y241A/W242A (O6YW/AA), FBXO27(O27), or FBXO27F262A/W263A (O27FW/AA), in combination with SKP1. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by blotting with the indicated antibodies and ConA lectin. ( B ) Localization of three glycoprotein-recognizing F-box proteins and FBXO27 mutants. HeLa cells stably expressing FBXO2-HA, FBXO6-HA, FBXO27-HA, the N-myristoylation defective mutant (FBXO27G2M-HA), or glycoprotein-binding–defective mutant (FBXO27FW/AA-HA) were subjected to immunostaining with anti-HA antibody. (Scale bars, 20 μm.) ( C ) The amino acid sequence of the N-terminal region of each F-box protein. The residues that constitute the N-myristoylation consensus sequence are aligned, and the predicted myristoylated Gly is boxed in red. ( D ) Detection of N-myristoylation in FBXO27. 293T cells expressing FBXO27-FLAG or FBXO27G2M-FLAG were incubated in the presence of myristic acid azide. Cell lysates were reacted with biotin alkyne, and FLAG-tagged proteins and biotin-labeled proteins were isolated with either anti-FLAG antibody or SA-agarose. Captured proteins were blotted with anti-FLAG antibody and <t>SA-HRP.</t> Arrows show the positions of FBXO27-FLAG.
    Hrp Conjugated, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 346 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Thermo Fisher streptavadin hrp conjugate
    FBXO27 is localized to membranes via N-myristoylation. ( A ) FBXO27 binds to glycoproteins modified with high-mannose N -glycans and TFRC. 293T and HeLa cells were transfected with plasmids encoding either only the <t>FLAG</t> tag, or FLAG-tagged FBXO2 (O2), FBXO6 (O6), FBXO6Y241A/W242A (O6YW/AA), FBXO27(O27), or FBXO27F262A/W263A (O27FW/AA), in combination with SKP1. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by blotting with the indicated antibodies and ConA lectin. ( B ) Localization of three glycoprotein-recognizing F-box proteins and FBXO27 mutants. HeLa cells stably expressing FBXO2-HA, FBXO6-HA, FBXO27-HA, the N-myristoylation defective mutant (FBXO27G2M-HA), or glycoprotein-binding–defective mutant (FBXO27FW/AA-HA) were subjected to immunostaining with anti-HA antibody. (Scale bars, 20 μm.) ( C ) The amino acid sequence of the N-terminal region of each F-box protein. The residues that constitute the N-myristoylation consensus sequence are aligned, and the predicted myristoylated Gly is boxed in red. ( D ) Detection of N-myristoylation in FBXO27. 293T cells expressing FBXO27-FLAG or FBXO27G2M-FLAG were incubated in the presence of myristic acid azide. Cell lysates were reacted with biotin alkyne, and FLAG-tagged proteins and biotin-labeled proteins were isolated with either anti-FLAG antibody or SA-agarose. Captured proteins were blotted with anti-FLAG antibody and <t>SA-HRP.</t> Arrows show the positions of FBXO27-FLAG.
    Streptavadin Hrp Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protein a hpr conjugate
    FBXO27 is localized to membranes via N-myristoylation. ( A ) FBXO27 binds to glycoproteins modified with high-mannose N -glycans and TFRC. 293T and HeLa cells were transfected with plasmids encoding either only the <t>FLAG</t> tag, or FLAG-tagged FBXO2 (O2), FBXO6 (O6), FBXO6Y241A/W242A (O6YW/AA), FBXO27(O27), or FBXO27F262A/W263A (O27FW/AA), in combination with SKP1. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by blotting with the indicated antibodies and ConA lectin. ( B ) Localization of three glycoprotein-recognizing F-box proteins and FBXO27 mutants. HeLa cells stably expressing FBXO2-HA, FBXO6-HA, FBXO27-HA, the N-myristoylation defective mutant (FBXO27G2M-HA), or glycoprotein-binding–defective mutant (FBXO27FW/AA-HA) were subjected to immunostaining with anti-HA antibody. (Scale bars, 20 μm.) ( C ) The amino acid sequence of the N-terminal region of each F-box protein. The residues that constitute the N-myristoylation consensus sequence are aligned, and the predicted myristoylated Gly is boxed in red. ( D ) Detection of N-myristoylation in FBXO27. 293T cells expressing FBXO27-FLAG or FBXO27G2M-FLAG were incubated in the presence of myristic acid azide. Cell lysates were reacted with biotin alkyne, and FLAG-tagged proteins and biotin-labeled proteins were isolated with either anti-FLAG antibody or SA-agarose. Captured proteins were blotted with anti-FLAG antibody and <t>SA-HRP.</t> Arrows show the positions of FBXO27-FLAG.
    Protein A Hpr Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher conjugated streptavidin
    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by <t>PE-streptavidin</t> and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P
    Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher avidin conjugated to hrp
    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by <t>PE-streptavidin</t> and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P
    Avidin Conjugated To Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated neutravidin
    Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse <t>IgG-HRP</t> or <t>NeutrAvidin</t> ® -HRP.
    Hrp Conjugated Neutravidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hrp conjugated avidin
    Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse <t>IgG-HRP</t> or <t>NeutrAvidin</t> ® -HRP.
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    Immunogenic proteins present on the 2-DE immunoblotted nitrocellulose membrane. All serum samples of patients and controls were subjected to 2-DE and blotted onto the nitrocellulose membrane prior to probing with pooled sera and monoclonal anti-human <t>IgM-HRP.</t>
    Hrp Polymer Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunogenic proteins present on the 2-DE immunoblotted nitrocellulose membrane. All serum samples of patients and controls were subjected to 2-DE and blotted onto the nitrocellulose membrane prior to probing with pooled sera and monoclonal anti-human <t>IgM-HRP.</t>
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    Thermo Fisher hrp conjugated antimouse
    Immunogenic proteins present on the 2-DE immunoblotted nitrocellulose membrane. All serum samples of patients and controls were subjected to 2-DE and blotted onto the nitrocellulose membrane prior to probing with pooled sera and monoclonal anti-human <t>IgM-HRP.</t>
    Hrp Conjugated Antimouse, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The IgG4 responses to O. volvulus -specific antigen (OvAg) in correlation with the age (in years) of the study participants.

    Journal: Parasite Epidemiology and Control

    Article Title: Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis infections in rural populations in central and southern Togo

    doi: 10.1016/j.parepi.2018.03.001

    Figure Lengend Snippet: The IgG4 responses to O. volvulus -specific antigen (OvAg) in correlation with the age (in years) of the study participants.

    Article Snippet: 5 μg/ml) in PBS pH 7.4 overnight, then the coating antigen solutions were discarded, and plates were blocked with PBS-Tween20® containing 5% fetal bovine serum at room temperature for 1.5 h. Thereafter, plates were washed with PBS-Tween20® (Sigma P3563), eluted blood samples were added without dilution and plates incubated at 37 °C for 2 h. After using PBS-Tween20® to wash an anti-human IgG4, horse radish peroxidase conjugated monoclonal antibody (Thermo Fisher Scientific, no.A10654) was added (dilution 1:500) for 1.5 h, then plates were washed as above and TMB substrate (Thermo Scientific 34021) was added.

    Techniques:

    The IgG4 responses to S. stercoralis L3 antigen (SsL3Ag) in correlation with the age (in years) of the study participants (upper graph), and the correlation of the SsL3Ag-specific IgG4 and the OvAg-specific IgG4 reactivity (lower graph) (OD = optical densities).

    Journal: Parasite Epidemiology and Control

    Article Title: Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis infections in rural populations in central and southern Togo

    doi: 10.1016/j.parepi.2018.03.001

    Figure Lengend Snippet: The IgG4 responses to S. stercoralis L3 antigen (SsL3Ag) in correlation with the age (in years) of the study participants (upper graph), and the correlation of the SsL3Ag-specific IgG4 and the OvAg-specific IgG4 reactivity (lower graph) (OD = optical densities).

    Article Snippet: 5 μg/ml) in PBS pH 7.4 overnight, then the coating antigen solutions were discarded, and plates were blocked with PBS-Tween20® containing 5% fetal bovine serum at room temperature for 1.5 h. Thereafter, plates were washed with PBS-Tween20® (Sigma P3563), eluted blood samples were added without dilution and plates incubated at 37 °C for 2 h. After using PBS-Tween20® to wash an anti-human IgG4, horse radish peroxidase conjugated monoclonal antibody (Thermo Fisher Scientific, no.A10654) was added (dilution 1:500) for 1.5 h, then plates were washed as above and TMB substrate (Thermo Scientific 34021) was added.

    Techniques:

    3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a HisProbe-HRP Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.

    Journal: Nature Communications

    Article Title: A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    doi: 10.1038/ncomms12780

    Figure Lengend Snippet: 3I14 blocks trypsin-mediated HA maturation and pH-dependent conformational changes. ( a ) Trypsin Cleavage Inhibition Assay. 0.4 μg recombinant H3-histidine (H3-BR07) was incubated in the presence of 2.5 μg 3I14 or Fm-6 IgG1, or in the absence of antibody in Tris-HCl buffer at pH 8.0 containing 2 μg ml −1 Trypsin at 37 °C. Trypsin digestion was stopped at several time-points by boiling the sample in a 100 °C water bath. Samples were run on 10% reduced SDS–PAGE and blotted using a HisProbe-HRP Abs. Data represent a representative experiment from three independent experiments. ( b ) 3I14 IgG1 prevented by low-pH triggered conformational rearrangements on the surface-expressed H3-A2/68 and H3-BR07. Upper panels show four various conformations of HA: uncleaved precursor (HA0, left); trypsin in neutral pH cleaved (mature HA, left middle); fusion pH cleaved (mature HA, right middle) and trimeric HA2 (HA2, right). The conformation rearrangements of surface-expressed H3 were detected by FACS staining of 3I14 (solid bars) and the head binding control mAb E730 (open bars). Binding is expressed as the percentage of binding to untreated HA (HA0). For this antibody inhibition assay, H3 was pretreated without mAb, with 3I14, or with control Ab, Fm-6 IgG1 before exposure of the cleaved HAs to pH 4.9. Data represent mean+s.d. of three independent experiments. SDS–PAGE, SDS–polyacrylamide electrophoresis.

    Article Snippet: Samples were run on 10% SDS–polyacrylamide electrophoresis gel under reducing conditions and blotted using a HisProbe-horseradish peroxidase and SuperSignal West HisProbe Kit (Pierce Biotechnology, Rockford, IL).

    Techniques: Inhibition, Recombinant, Incubation, SDS Page, FACS, Staining, Binding Assay, Electrophoresis

    Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with Neutravidin-HRP conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.

    Journal: Biochemistry

    Article Title: Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor

    doi: 10.1021/bi802061z

    Figure Lengend Snippet: Immunoblot analysis of Bio-DOPA-α-factor reacted with membranes lacking Ste2p. Cell membranes containing and lacking Ste2p were treated with Bio-DOPA-α-factor for cross-linking. The samples were separated on SDS-PAGE and probed with an antibody directed against Ste2p ( A ) and with Neutravidin-HRP conjugate ( B ). M indicates membrane extract, P indicates enriched samples from His-HC Nickel resin.

    Article Snippet: The blots were probed with an antibody directed against the N-terminal 60 amino acids of Ste2p [generously provided by J. Konopka ( )] and with Neutravidin-HRP conjugate (Pierce, Rockford, IL) to detect the biotin tag on Bio-DOPA pheromone covalently linked to the Ste2p.

    Techniques: SDS Page

    Bio-DOPA-α-factor chemical cross-linking into Ste2p. Cell membranes containing Ste2p were incubated with Bio-DOPA-α-factor alone or with Bio-DOPA-α-factor in the presence of 100-fold wild-type α-factor, antagonist, synergist, or BSA. The membranes were treated with sodium periodate to oxidize DOPA and initiate cross-linking, and the reaction was quenched with DTT. The samples were resolved on SDS-PAGE and probed with an antibody against Ste2p ( A ) and with Neutravidin-HRP conjugate to detect the biotin tag on Bio-DOPA-α-factor ( B ).

    Journal: Biochemistry

    Article Title: Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor

    doi: 10.1021/bi802061z

    Figure Lengend Snippet: Bio-DOPA-α-factor chemical cross-linking into Ste2p. Cell membranes containing Ste2p were incubated with Bio-DOPA-α-factor alone or with Bio-DOPA-α-factor in the presence of 100-fold wild-type α-factor, antagonist, synergist, or BSA. The membranes were treated with sodium periodate to oxidize DOPA and initiate cross-linking, and the reaction was quenched with DTT. The samples were resolved on SDS-PAGE and probed with an antibody against Ste2p ( A ) and with Neutravidin-HRP conjugate to detect the biotin tag on Bio-DOPA-α-factor ( B ).

    Article Snippet: The blots were probed with an antibody directed against the N-terminal 60 amino acids of Ste2p [generously provided by J. Konopka ( )] and with Neutravidin-HRP conjugate (Pierce, Rockford, IL) to detect the biotin tag on Bio-DOPA pheromone covalently linked to the Ste2p.

    Techniques: Incubation, SDS Page

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

    Journal: BMC Cell Biology

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

    doi: 10.1186/s12860-014-0045-1

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

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

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

    Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Journal: Scientific Reports

    Article Title: Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

    doi: 10.1038/s41598-018-20527-6

    Figure Lengend Snippet: Correlation between apoptotic signaling and the generation of large DNAs with decreased generation of excised oligonucleotide repair products. ( A ) Analysis of large DNA fragments and excised oligomers released from genomic DNA following UV irradiation. HeLa cells were exposed to 20 J/m 2 of UVC and incubated for the indicated time points. After extraction of low molecular weight of DNAs from the cells, the DNA molecules were labeled with biotin at 3′-end using terminal deoxynucleotidyl transferase (TdT). The labeled DNA molecules were separated by gel electrophoresis, transferred to a nylon membrane and immobilized by UV cross-linking. The membrane was then incubated with HRP-conjugated streptavidin, and the labeled DNA molecules were detected with a chemiluminescence reagent (upper panel). A small portion of the UV-irradiated cells were lysed and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. ( B ) Quantification of the excised oligonucleotide repair products and cleaved PARP signals as shown in ( A ). The relative signal intensities were determined by setting the highest signal to 100. The results were quantified and are plotted as mean and standard deviations. ( C ) Quantitative analysis of the excised repair products and the large DNA molecules. ( D ) Wild-type (AA8) and XPG mutant (UV135) CHO cell lines were exposed to UV and processed as described above for the detection of soluble DNAs.

    Article Snippet: The membrane was incubated with 1X PBS containing 2% SDS for 30 min and incubated with streptavidin-HRP conjugates (Thermo Scientific) in 1× PBS containing 2% SDS for 30 min.

    Techniques: Irradiation, Incubation, Molecular Weight, Labeling, Nucleic Acid Electrophoresis, SDS Page, Mutagenesis

    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: Twenty μg of total protein was boiled for 2 min in SDS sample buffer, resolved by 10% SDS-PAGE gel with molecular weight standards (Precision Plus Protein™ All Blue Standards, Bio-Rad), and transferred onto nitrocellulose membranes., The membranes were blocked with 5% bovine serum albumin (BSA) fraction V (Invitrogen, Grand Island, NY) and immunoblotted with streptavidin-HRP conjugate (Pierce, Rockford, IL), anti-Gluc (rabbit; Nanolight, Pinetop, AZ) and anti-GAPDH antibodies (mouse; Calbiochem, San Diego, CA).

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

    Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Journal: Cell Cycle

    Article Title: AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

    doi: 10.1080/15384101.2017.1310350

    Figure Lengend Snippet: Identification of in vivo binding partners of AKAP95. (A) Schematic representation of Myc-BirA*-AKAP95. (B) Western blot analysis using anti-myc antibody and HRP-labeled streptavidin of lysates from Myc-BirA*-AKAP95 expressing- and control U2OS cells cultured as indicated. (C) Immunolocalization of Myc-BirA*-AKAP95 (anti-myc; red) and protein biotinylation (FITC-labeled streptavidin; green) in stable Myc-BirA*–AKAP95 U2OS cells cultured as indicated. Immunolocalization of AKAP95 in U2OS cells (bottom). Scale bar, 5 μm. (D) Gene ontology analysis and nuclear component classification of proteins identified by AKAP95 BioID. (E) Western blot analysis using indicated antibodies of the samples from Myc-BirA*-AKAP95-expressing and control U2OS cells that were subjected to BioID.

    Article Snippet: Biotinylated proteins were detected directly using HRP-Streptavidin conjugate (Invitrogen; 43-4323; 1:5000).

    Techniques: In Vivo, Binding Assay, Western Blot, Labeling, Expressing, Cell Culture

    FBXO27 is localized to membranes via N-myristoylation. ( A ) FBXO27 binds to glycoproteins modified with high-mannose N -glycans and TFRC. 293T and HeLa cells were transfected with plasmids encoding either only the FLAG tag, or FLAG-tagged FBXO2 (O2), FBXO6 (O6), FBXO6Y241A/W242A (O6YW/AA), FBXO27(O27), or FBXO27F262A/W263A (O27FW/AA), in combination with SKP1. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by blotting with the indicated antibodies and ConA lectin. ( B ) Localization of three glycoprotein-recognizing F-box proteins and FBXO27 mutants. HeLa cells stably expressing FBXO2-HA, FBXO6-HA, FBXO27-HA, the N-myristoylation defective mutant (FBXO27G2M-HA), or glycoprotein-binding–defective mutant (FBXO27FW/AA-HA) were subjected to immunostaining with anti-HA antibody. (Scale bars, 20 μm.) ( C ) The amino acid sequence of the N-terminal region of each F-box protein. The residues that constitute the N-myristoylation consensus sequence are aligned, and the predicted myristoylated Gly is boxed in red. ( D ) Detection of N-myristoylation in FBXO27. 293T cells expressing FBXO27-FLAG or FBXO27G2M-FLAG were incubated in the presence of myristic acid azide. Cell lysates were reacted with biotin alkyne, and FLAG-tagged proteins and biotin-labeled proteins were isolated with either anti-FLAG antibody or SA-agarose. Captured proteins were blotted with anti-FLAG antibody and SA-HRP. Arrows show the positions of FBXO27-FLAG.

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

    Article Title: Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

    doi: 10.1073/pnas.1702615114

    Figure Lengend Snippet: FBXO27 is localized to membranes via N-myristoylation. ( A ) FBXO27 binds to glycoproteins modified with high-mannose N -glycans and TFRC. 293T and HeLa cells were transfected with plasmids encoding either only the FLAG tag, or FLAG-tagged FBXO2 (O2), FBXO6 (O6), FBXO6Y241A/W242A (O6YW/AA), FBXO27(O27), or FBXO27F262A/W263A (O27FW/AA), in combination with SKP1. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-FLAG antibody, and analyzed by blotting with the indicated antibodies and ConA lectin. ( B ) Localization of three glycoprotein-recognizing F-box proteins and FBXO27 mutants. HeLa cells stably expressing FBXO2-HA, FBXO6-HA, FBXO27-HA, the N-myristoylation defective mutant (FBXO27G2M-HA), or glycoprotein-binding–defective mutant (FBXO27FW/AA-HA) were subjected to immunostaining with anti-HA antibody. (Scale bars, 20 μm.) ( C ) The amino acid sequence of the N-terminal region of each F-box protein. The residues that constitute the N-myristoylation consensus sequence are aligned, and the predicted myristoylated Gly is boxed in red. ( D ) Detection of N-myristoylation in FBXO27. 293T cells expressing FBXO27-FLAG or FBXO27G2M-FLAG were incubated in the presence of myristic acid azide. Cell lysates were reacted with biotin alkyne, and FLAG-tagged proteins and biotin-labeled proteins were isolated with either anti-FLAG antibody or SA-agarose. Captured proteins were blotted with anti-FLAG antibody and SA-HRP. Arrows show the positions of FBXO27-FLAG.

    Article Snippet: The pull-down products and immune complexes were eluted by heating in SDS/PAGE sample buffer, and were subjected to immunoblot analyses by anti-FLAG antibody and HRP-conjugated SA (Pierce).

    Techniques: Modification, Transfection, FLAG-tag, Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis, Binding Assay, Immunostaining, Sequencing, Incubation, Labeling, Isolation

    Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by PE-streptavidin and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P

    Journal: Journal of Leukocyte Biology

    Article Title: CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells

    doi: 10.1189/jlb.2A0414-193RR

    Figure Lengend Snippet: Anti-CCR5 mAb binding to human blood cells and CHO–CCR5 transfectants. (A) Diagram mapping the different CCR5 epitopes recognized by monoclonal antibodies used in our study. (B–C) Anti-CCR5 mAbs binding experiments performed on human monocytes, MDMs, and T cell blasts labeled live with a 5 µg/ml concentration of each anti-CCR5 mAb. Cell-bound antibodies were detected with biotin-conjugated secondary antibody followed by PE-streptavidin and cell-associated fluorescent signal measured by flow cytometry. (B) Box and whisker plots of isotype-corrected MFI values, showing the range of antibody-binding levels on cells derived from different donors ( N = 7). (C) Cells derived from the same donors show a significant increase in MC5, CTC5, and 2D7 binding after differentiation of blood monocytes into MDMs ( N = 11). * P ≤ 0.05 *** P ≤ 0.01 paired Student’s t test. (D) Like blood cells, CHO-CCR5 cells were labeled live with the different anti-CCR5 mAbs, but cell-bound antibodies were detected with a PE-conjugated secondary antibody; the graph plots the isotype-corrected MFI values (means ± sd ) from a representative triplicate experiment. (E) Compared binding curves of each antibody for CHO-CCR5 cells, T cell blasts, and MDMs; results are normalized to the MFI of the highest antibody concentration and represent the means ± sd of N = 3 independent, triplicate experiments. * P

    Article Snippet: Reagents and antibodies Tissue-culture reagents, all secondary antibodies, and conjugated-streptavidin were purchased from Thermo Fisher Scientific (Paisley, Renfrewshire, United Kingdom).

    Techniques: Binding Assay, Labeling, Concentration Assay, Flow Cytometry, Cytometry, Whisker Assay, Derivative Assay

    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

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

    Journal: Biochemistry

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

    doi: 10.1021/bi4014825

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

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

    Techniques: Labeling, Incubation

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

    Journal: Nature Communications

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

    doi: 10.1038/ncomms7765

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

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

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

    Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse IgG-HRP or NeutrAvidin ® -HRP.

    Journal: The journal of gene medicine

    Article Title: Gene delivery in malignant B cells using the combination of lentiviruses conjugated to anti-transferrin receptor antibodies and an immunoglobulin promoter

    doi: 10.1002/jgm.2754

    Figure Lengend Snippet: Targeting strategies and verification of pseudotyped viral coat proteins Schematic representation of viruses conjugated to A) ch128.1 through 2.2 SINDBIS and B) ch128.1Av through biotin-avidin interaction with BAP SINDBIS. C) Western blot analysis of chimeric Sindbis virus envelope proteins. Lentivirus pseudotyped with 2.2 SINDBIS or BAP SINDBIS produced in the presence of pSec BirA and biotin were analyzed using anti-Sindbis ascitic fluid and a secondary anti-mouse IgG-HRP or NeutrAvidin ® -HRP.

    Article Snippet: Biotinylation of envelope proteins was detected using HRP-conjugated NeutrAvidin® (Thermo Fisher Scientific).

    Techniques: Avidin-Biotin Assay, Western Blot, Produced

    Immunogenic proteins present on the 2-DE immunoblotted nitrocellulose membrane. All serum samples of patients and controls were subjected to 2-DE and blotted onto the nitrocellulose membrane prior to probing with pooled sera and monoclonal anti-human IgM-HRP.

    Journal: PeerJ

    Article Title: Identification of biomarkers for periodontal disease using the immunoproteomics approach

    doi: 10.7717/peerj.2327

    Figure Lengend Snippet: Immunogenic proteins present on the 2-DE immunoblotted nitrocellulose membrane. All serum samples of patients and controls were subjected to 2-DE and blotted onto the nitrocellulose membrane prior to probing with pooled sera and monoclonal anti-human IgM-HRP.

    Article Snippet: The membranes were then incubated with monoclonal anti-human IgM conjugated to horseradish peroxidase (HRP) (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:6,000 for 1 h at room temperature.

    Techniques: