hrp conjugate Millipore Search Results


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  • 99
    Thermo Fisher hrp conjugated secondary antibodies
    Hrp Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6418 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hrp conjugate secondary antibody millipore
    Hrp Conjugate Secondary Antibody Millipore, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hrp streptavidin
    IEC-6 cell lysate was incubated with biotin-CLEFMA (10 and 20 μM) or CLEFMA-N-biotin (10 μM) and separated on a gel. After transferring to a membrane, the proteins were probed <t>HRP-streptavidin.</t>
    Hrp Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hrp
    Co-immunoprecipitation assay (Co-IP). (A) Location of residue K265 in the 3D crystal structure of ZIKV NS1. (B) Flowchart of Co-IP experiments from ZIKV infected cells. Rabbit IgG anti-NS1 or control <t>IgGs</t> were used for pull down ZIKV NS1 and its associated complexes. (C) Co-IP results from (B). NS1 in both eluates and cell lysates were probed using rabbit anti-NS1 and protein <t>A-HRP.</t> E proteins were detected by mouse IgG anti-ZIKV and goat anti-mouse IgG-HRP. prM was examined by rabbit IgG anti-ZIKV prM and goat anti-rabbit IgG-HRP. (D) Densitometry analysis of Western blot results from (C). The band intensities of prM and E proteins in (C) were quantified and normalized to those of NS1 proteins from corresponding eluates. The efficiencies of prM and E pulled-down by WT NS1 were set as 100%. The averaged relative intensities from three independent experiments were shown. An unpaired Student t- test was used to estimate the statistical significance. (E) Flowchart of Co-IP from HEK293T cells transiently expressing NS1 and NS2A-HA. Cell lysates was subjected to Co-IP using mouse IgG anti-HA or mouse control IgG. (F) Western blot results from (E). NS2A-HA in the eluates and cell lysates were examined by rabbit IgG anti-HA and goat anti-rabbit IgG-HRP respectively. NS1 proteins were detected by rabbit anti-ZIKV NS1 and goat anti-rabbit IgG-HRP. (G) Densitometry analysis of Western blot results from (F). The band intensities of NS1 proteins in (F) were quantified and normalized to those of NS2A-HA proteins from corresponding eluates. An unpaired Student t- test was used to evaluate the statistical significance.
    Hrp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore streptavidin hrp
    Determination of GP1/PS ratios in rLCMV-LASVGP and authentic LASV. (A) Schematic of the capture ELISA. Virus is captured by immobilized MAb 86.3, which recognizes a conserved nonneutralizing epitope in GP2. After removal of unbound material, bound virus is probed with ANX-V to detect PS, displayed in the lipid bilayer of the viral envelope, and DGEKFc4, which binds to GP1. For details, please see the text. (B) Detection of GP1 and PS. Purified rLCMV-LASVGP and authentic inactivated LASV were incubated with immobilized MAb 83.6, with the nonenveloped AdV5-GFP used as a negative control. After the plates were washed, bound virus was incubated with biotin-conjugated ANX-V in the presence and absence of EGTA, as well as DGEKFc4. Bound biotinylated ANX-V and DGEKFc4 were detected by <t>streptavidin-HRP-</t> and HRP-conjugated anti-human Fc antibody in a color reaction. Please note the reduced ANX-V binding in the presence of the Ca 2+ chelator EGTA. Data are means ± SD ( n = 3). (C) Ratios of GP1 to PS calculated for the virus preparations tested in panel B. OD (450), optical density at 450 nm; LASVi, inactivated LASV.
    Streptavidin Hrp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore horseradish peroxide conjugate
    Determination of GP1/PS ratios in rLCMV-LASVGP and authentic LASV. (A) Schematic of the capture ELISA. Virus is captured by immobilized MAb 86.3, which recognizes a conserved nonneutralizing epitope in GP2. After removal of unbound material, bound virus is probed with ANX-V to detect PS, displayed in the lipid bilayer of the viral envelope, and DGEKFc4, which binds to GP1. For details, please see the text. (B) Detection of GP1 and PS. Purified rLCMV-LASVGP and authentic inactivated LASV were incubated with immobilized MAb 83.6, with the nonenveloped AdV5-GFP used as a negative control. After the plates were washed, bound virus was incubated with biotin-conjugated ANX-V in the presence and absence of EGTA, as well as DGEKFc4. Bound biotinylated ANX-V and DGEKFc4 were detected by <t>streptavidin-HRP-</t> and HRP-conjugated anti-human Fc antibody in a color reaction. Please note the reduced ANX-V binding in the presence of the Ca 2+ chelator EGTA. Data are means ± SD ( n = 3). (C) Ratios of GP1 to PS calculated for the virus preparations tested in panel B. OD (450), optical density at 450 nm; LASVi, inactivated LASV.
    Horseradish Peroxide Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti gst hrp
    NFATC3 selectively binds to IRF7 and enhances its transcriptional activity. (A) Fresh isolated human primary pDCs or pDCs stimulated with CpG A for 4 h. Cells were fixed with paraformaldehyde, permeablized, stained with anti-IRF7 (green) and anti-NFATC3 (red), and mounted with Prolong gold antifade reagent with DAPI (blue). Cells were analyzed using confocal microscopy. (B) Untouched human pDCs were isolated as described in the Confocal microscopy section of Materials and methods. pDCs were left untreated or treated with 1 µM CpG A for 3 h, and pDCs were lysed and immunoprecipitated (IP) with NFATC3 antibody. IRF7 was detected by immunoblotting (IB). (C) Purified <t>GST</t> or GST-tagged NFATC3 were incubated with Myc-tagged IRF7. Myc-tagged IRF7 was immunoprecipitated with Myc–agarose beads. GST was detected by anti-GST <t>HRP</t> conjugate antibody. (D) Purified Myc-tagged IRF3 or IRF7 was incubated with GST-tagged NFATC3, and GST-tagged NFATC3 was immunoprecipitated with glutathione Sepharose beads. Then, Myc-tagged proteins were detected by anti-Myc antibody. (E) HEK-293T cells were transfected with HA-tagged NFAT plasmids and Myc-tagged IRF7; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (F) HEK-293T cells were transfected with HA-tagged NFATC3 plasmids and Myc-tagged MyD88, IRF3, and IRF7 plasmids; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (G and H) HEK-293T cells were transfected with 100 ng IFN-α4 luciferase reporter plasmid (IFNα4-Luc) or IFN-β luciferase reporter plasmid (IFNβ-Luc) and 0.5 ng IRF7 expression vector together with an increasing amount of NFATC3 expression vector (25, 50, and 100 ng). 0.1 ng renilla luciferase reporter plasmid was transfected simultaneously as an internal control. Results are presented as fold induction relative to the activity of renilla luciferase. (I) HEK-293T cells were transfected with 100 ng NF-κB luciferase reporter plasmid (NFκB-Luc) together with 5 ng MyD88 expression vector or an increasing amount of NFATC3 expression vector. (J) HEK-293T cells were transfected with 100 ng IFN-β luciferase reporter plasmid (IFNβ-Luc) together with 5 ng IKKi expression vector or an increasing amount of NFATC3 expression vector. (G–J) Data are representative of four independent experiments.
    Anti Gst Hrp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore m2 hrp conjugate
    NFATC3 selectively binds to IRF7 and enhances its transcriptional activity. (A) Fresh isolated human primary pDCs or pDCs stimulated with CpG A for 4 h. Cells were fixed with paraformaldehyde, permeablized, stained with anti-IRF7 (green) and anti-NFATC3 (red), and mounted with Prolong gold antifade reagent with DAPI (blue). Cells were analyzed using confocal microscopy. (B) Untouched human pDCs were isolated as described in the Confocal microscopy section of Materials and methods. pDCs were left untreated or treated with 1 µM CpG A for 3 h, and pDCs were lysed and immunoprecipitated (IP) with NFATC3 antibody. IRF7 was detected by immunoblotting (IB). (C) Purified <t>GST</t> or GST-tagged NFATC3 were incubated with Myc-tagged IRF7. Myc-tagged IRF7 was immunoprecipitated with Myc–agarose beads. GST was detected by anti-GST <t>HRP</t> conjugate antibody. (D) Purified Myc-tagged IRF3 or IRF7 was incubated with GST-tagged NFATC3, and GST-tagged NFATC3 was immunoprecipitated with glutathione Sepharose beads. Then, Myc-tagged proteins were detected by anti-Myc antibody. (E) HEK-293T cells were transfected with HA-tagged NFAT plasmids and Myc-tagged IRF7; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (F) HEK-293T cells were transfected with HA-tagged NFATC3 plasmids and Myc-tagged MyD88, IRF3, and IRF7 plasmids; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (G and H) HEK-293T cells were transfected with 100 ng IFN-α4 luciferase reporter plasmid (IFNα4-Luc) or IFN-β luciferase reporter plasmid (IFNβ-Luc) and 0.5 ng IRF7 expression vector together with an increasing amount of NFATC3 expression vector (25, 50, and 100 ng). 0.1 ng renilla luciferase reporter plasmid was transfected simultaneously as an internal control. Results are presented as fold induction relative to the activity of renilla luciferase. (I) HEK-293T cells were transfected with 100 ng NF-κB luciferase reporter plasmid (NFκB-Luc) together with 5 ng MyD88 expression vector or an increasing amount of NFATC3 expression vector. (J) HEK-293T cells were transfected with 100 ng IFN-β luciferase reporter plasmid (IFNβ-Luc) together with 5 ng IKKi expression vector or an increasing amount of NFATC3 expression vector. (G–J) Data are representative of four independent experiments.
    M2 Hrp Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore streptavidin horseradish peroxidase hrp conjugate
    The specific interactions between Cry6Aa and ASP-1. (A) The expression and purification of ASP1-GST fusion protein in E . coli . Lane 1, E . coli BL21 containing pGEX-6p-1 vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet; lane 2, E . coli BL21 containing pASP1-GSTvector without IPTG induction, cell pellet. lane 3, E . coli BL21 containing pASP1-GST vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet. lane 4, purified ASP1-GST fusion protein eluted from the glutathione Sepharose bulk; lane 5, purified GST tag eluted from the glutathione Sepharose bulk. (B) The binding of Cry6Aa to ASP1-GST fusion proteins by ligand blotting. The purified ASP1-GST fusion proteins (lane 6) or GST (lane 7) were separated by SDS-PAGE gels, and were transferred to a PVDF membrane. Filters were blocked overnight, and then probed with biotinylated Cry6Aa, Unbound toxin was removed by washing. The bound protein was detected <t>streptavidin-horseradish</t> peroxidase <t>(HRP)</t> conjugate. And finally the membrane was visualized. GST was the control. (C) ASP-1 proteins were dotted on a NC membrane directly and were probed with biotin labeled Cry6Aa or with biotin labeled Cry6Aa plus unlabeled Cry6Aa (1000-fold). GST was the control. (D) Binding affinity of Cry6Aa to ASP-1 was determined by ELISA. Ninety-six-well microtiter plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry6Aa alone or with 1000-fold molar excess of unlabeled Cry6Aa to determine specific binding. Each point represents mean amounts of protein specifically bound. Specific binding was determined by subtracting nonspecific binding (with 1000-fold molar excess of unlabeled Cry6Aa) from total binding (without excess unlabeled Cry6Aa). (E) Isothermal titration calorimetric analysis of Cry6Aa binding to ASP-1. Titration of ASP-1 (2.20 μM) with 21.16 μM Cry6Aa. The top panel show the raw data of the heat released, and the bottom panel show the binding isotherm fitted using nonlinear binding models. Data were analyzed in Origin 8.6 software after subtracting the heat released from titrating Cry6Aa alone into buffer. One of three representative experiments is shown.
    Streptavidin Horseradish Peroxidase Hrp Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore hrp avidin conjugate
    Affinity precipitation (A) and blot overlay (B) analysis of the interaction between palladin and ezrin. (A) Yeast lysates expressing HA-tagged palladin Ig1, Ig2–3, and ezrin 1–309 were incubated with glutathione-Sepharose–coupled GST-ezrin 279–531, GST-ezrin 477–585, and GST. After washes bound material was eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters. Proteins were detected by HA-immunoblotting with the use of 12CA5 mAb. Palladin Ig2–3 interacts with the α-helical domain of ezrin (279–531) but not with the C-terminal domain (477–585). As a positive control, the association of ezrin 1–309 and ezrin 477–585 is shown. (B) Total lysates of HISM and U251mg cells, purified ezrin, or recombinant ezrin 1–309 were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with biotinylated probes GST, GST-palladin Ig2–3, and GST-palladin Ig1–3. Bound probe was detected with the use of <t>HRP-conjugated</t> <t>extravidin</t> and enhanced chemiluminiscence. Both palladin probes bind to two (HISM) or three (U251 mg) major protein bands. Reprobing of the blots with ezrin antibody after stripping shows that the 75-kDa band recognized by palladin is consistent with being ezrin. The probes also react with immobilized purified ezrin but not with the N-terminal domain.
    Hrp Avidin Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore horse radish peroxidase hrp
    Affinity precipitation (A) and blot overlay (B) analysis of the interaction between palladin and ezrin. (A) Yeast lysates expressing HA-tagged palladin Ig1, Ig2–3, and ezrin 1–309 were incubated with glutathione-Sepharose–coupled GST-ezrin 279–531, GST-ezrin 477–585, and GST. After washes bound material was eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters. Proteins were detected by HA-immunoblotting with the use of 12CA5 mAb. Palladin Ig2–3 interacts with the α-helical domain of ezrin (279–531) but not with the C-terminal domain (477–585). As a positive control, the association of ezrin 1–309 and ezrin 477–585 is shown. (B) Total lysates of HISM and U251mg cells, purified ezrin, or recombinant ezrin 1–309 were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with biotinylated probes GST, GST-palladin Ig2–3, and GST-palladin Ig1–3. Bound probe was detected with the use of <t>HRP-conjugated</t> <t>extravidin</t> and enhanced chemiluminiscence. Both palladin probes bind to two (HISM) or three (U251 mg) major protein bands. Reprobing of the blots with ezrin antibody after stripping shows that the 75-kDa band recognized by palladin is consistent with being ezrin. The probes also react with immobilized purified ezrin but not with the N-terminal domain.
    Horse Radish Peroxidase Hrp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore s protein hrp conjugate
    <t>A27</t> viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat <t>anti-rabbit-HRP</t> antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p
    S Protein Hrp Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore flag m2 hrp conjugate
    <t>A27</t> viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat <t>anti-rabbit-HRP</t> antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p
    Flag M2 Hrp Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore hrp conjugate antibody
    <t>A27</t> viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat <t>anti-rabbit-HRP</t> antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p
    Hrp Conjugate Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore hrp conjugated extraevidin
    <t>A27</t> viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat <t>anti-rabbit-HRP</t> antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p
    Hrp Conjugated Extraevidin, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore hrp conjugated avidin
    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
    Hrp Conjugated Avidin, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore hrp conjugated ctxb
    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
    Hrp Conjugated Ctxb, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore hrp conjugated m2
    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
    Hrp Conjugated M2, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
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    Millipore hrp conjugated ap307p
    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
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    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with <t>HRP-conjugated</t> avidin. C: control. 1–7: patients 1–7. The α subunit of <t>3-methylcrotonyl-CoA</t> carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls
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    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    Millipore igg2a hrp conjugates
    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of <t>EXOG-FLAG-tagged</t> was monitored by Western analysis with <t>FLAG-HRP</t> conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
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    MAGE proteins interact with RING-containing proteins. ( A ) Summary of the MAGE-RING interaction results. MAGEA1(1–309) (A1), MAGEB1(63–315) (B1), MAGEC2(129–339) (C2), MAGEE2(87–505) (E2), MAGEF1(74–260) (F1) and/or MAGEL2(420–630) (L2) baits were used to screen Y2H library of RING-finger preys. The Y2H interactions are shown: black - high confidence Y2H interactions observed on both -His and -Ade selection; gray - low confidence interactions (from Table S1 ). Then, selected MAGE-RING interactions were tested using co-immunoprecipitation and pull-down assays: +++ means strong interaction; ++ means intermediate strength of interaction; + means weak interaction; - means no interaction (based on results shown in Figs. S1 , S2 and S4 ); ND – not determined. Nine out of 14 MAGE-RING interactions detected in Y2H were confirmed. ( B ) Two examples of the MAGE-RING co-immunoprecipitation results. His-S-tag-MAGEA1 (lanes 1–3 and 7–9) was co-transfected with either GFP-TRIM8 (lanes 1–6) or myc-TRIM31 (lanes 7–12) into HEK293T cells. The HEK293T cell extracts were incubated with <t>protein-S-beads</t> and immunoprecipitated proteins were analyzed on western blots. MAGEA1 was detected using <t>protein-S-HRP</t> conjugate and the RING-finger-containing proteins were visualized using anti-GFP and/or anti-myc antibody, respectively. In control experiments, the pTriEx4 vector was co-transfected with either GFP-TRIM8 (lanes 4–6) or myc-TRIM31 (lanes 10–12). The input (I, 1% of lysate), unbound (U, 1% of lysate) and bound (B, 10% of lysate) fractions were separated by 12% SDS-PAGE.
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    MAGE proteins interact with RING-containing proteins. ( A ) Summary of the MAGE-RING interaction results. MAGEA1(1–309) (A1), MAGEB1(63–315) (B1), MAGEC2(129–339) (C2), MAGEE2(87–505) (E2), MAGEF1(74–260) (F1) and/or MAGEL2(420–630) (L2) baits were used to screen Y2H library of RING-finger preys. The Y2H interactions are shown: black - high confidence Y2H interactions observed on both -His and -Ade selection; gray - low confidence interactions (from Table S1 ). Then, selected MAGE-RING interactions were tested using co-immunoprecipitation and pull-down assays: +++ means strong interaction; ++ means intermediate strength of interaction; + means weak interaction; - means no interaction (based on results shown in Figs. S1 , S2 and S4 ); ND – not determined. Nine out of 14 MAGE-RING interactions detected in Y2H were confirmed. ( B ) Two examples of the MAGE-RING co-immunoprecipitation results. His-S-tag-MAGEA1 (lanes 1–3 and 7–9) was co-transfected with either GFP-TRIM8 (lanes 1–6) or myc-TRIM31 (lanes 7–12) into HEK293T cells. The HEK293T cell extracts were incubated with <t>protein-S-beads</t> and immunoprecipitated proteins were analyzed on western blots. MAGEA1 was detected using <t>protein-S-HRP</t> conjugate and the RING-finger-containing proteins were visualized using anti-GFP and/or anti-myc antibody, respectively. In control experiments, the pTriEx4 vector was co-transfected with either GFP-TRIM8 (lanes 4–6) or myc-TRIM31 (lanes 10–12). The input (I, 1% of lysate), unbound (U, 1% of lysate) and bound (B, 10% of lysate) fractions were separated by 12% SDS-PAGE.
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    MAGE proteins interact with RING-containing proteins. ( A ) Summary of the MAGE-RING interaction results. MAGEA1(1–309) (A1), MAGEB1(63–315) (B1), MAGEC2(129–339) (C2), MAGEE2(87–505) (E2), MAGEF1(74–260) (F1) and/or MAGEL2(420–630) (L2) baits were used to screen Y2H library of RING-finger preys. The Y2H interactions are shown: black - high confidence Y2H interactions observed on both -His and -Ade selection; gray - low confidence interactions (from Table S1 ). Then, selected MAGE-RING interactions were tested using co-immunoprecipitation and pull-down assays: +++ means strong interaction; ++ means intermediate strength of interaction; + means weak interaction; - means no interaction (based on results shown in Figs. S1 , S2 and S4 ); ND – not determined. Nine out of 14 MAGE-RING interactions detected in Y2H were confirmed. ( B ) Two examples of the MAGE-RING co-immunoprecipitation results. His-S-tag-MAGEA1 (lanes 1–3 and 7–9) was co-transfected with either GFP-TRIM8 (lanes 1–6) or myc-TRIM31 (lanes 7–12) into HEK293T cells. The HEK293T cell extracts were incubated with <t>protein-S-beads</t> and immunoprecipitated proteins were analyzed on western blots. MAGEA1 was detected using <t>protein-S-HRP</t> conjugate and the RING-finger-containing proteins were visualized using anti-GFP and/or anti-myc antibody, respectively. In control experiments, the pTriEx4 vector was co-transfected with either GFP-TRIM8 (lanes 4–6) or myc-TRIM31 (lanes 10–12). The input (I, 1% of lysate), unbound (U, 1% of lysate) and bound (B, 10% of lysate) fractions were separated by 12% SDS-PAGE.
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    Image Search Results


    IEC-6 cell lysate was incubated with biotin-CLEFMA (10 and 20 μM) or CLEFMA-N-biotin (10 μM) and separated on a gel. After transferring to a membrane, the proteins were probed HRP-streptavidin.

    Journal: Frontiers in Chemistry

    Article Title: Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

    doi: 10.3389/fchem.2018.00392

    Figure Lengend Snippet: IEC-6 cell lysate was incubated with biotin-CLEFMA (10 and 20 μM) or CLEFMA-N-biotin (10 μM) and separated on a gel. After transferring to a membrane, the proteins were probed HRP-streptavidin.

    Article Snippet: Anti-actin primary antibody, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody, and HRP-streptavidin were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Incubation, Transferring

    Effect of CLEFMA on proteolytic stability of RPN13. Whole cell lysate of H441 cells was treated with biotin-CLEFMA, followed by thermolysin digestion at room temperature for 10–60 min. The mixture was separated on an acrylamide gel, and the separated proteins were subjected to (A) Coomassie Brilliant staining, (B) HRP-Streptavidin probing for chemiluminescence imaging, and (C) Anti-RPN13 immunoblotting.

    Journal: Frontiers in Chemistry

    Article Title: Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

    doi: 10.3389/fchem.2018.00392

    Figure Lengend Snippet: Effect of CLEFMA on proteolytic stability of RPN13. Whole cell lysate of H441 cells was treated with biotin-CLEFMA, followed by thermolysin digestion at room temperature for 10–60 min. The mixture was separated on an acrylamide gel, and the separated proteins were subjected to (A) Coomassie Brilliant staining, (B) HRP-Streptavidin probing for chemiluminescence imaging, and (C) Anti-RPN13 immunoblotting.

    Article Snippet: Anti-actin primary antibody, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody, and HRP-streptavidin were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Acrylamide Gel Assay, Staining, Imaging

    CLEFMA and EF24 interact with RPN13 in the fully assembled 26S proteasome and whole cell lysate. (A) The biotinylated compounds were allowed to react with purified human 26S proteasome, the mixture was separated on poly-acrylamide gels, and the transfer membrane was probed with HRP-streptavidin (blot #1). The blot was stripped and re-probed with anti-human RPN13 (blot #2). (B) Streptavidin bead-mediated pull down of RPN13 from rat IEC-6 cell lysate treated with biotinylated EF24 or CLEFMA. After blotting with anti-human HRP-streptavidin (blot #3), the membrane was stripped and re-probed with anti-rat RPN13 antibody (blot #4).

    Journal: Frontiers in Chemistry

    Article Title: Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

    doi: 10.3389/fchem.2018.00392

    Figure Lengend Snippet: CLEFMA and EF24 interact with RPN13 in the fully assembled 26S proteasome and whole cell lysate. (A) The biotinylated compounds were allowed to react with purified human 26S proteasome, the mixture was separated on poly-acrylamide gels, and the transfer membrane was probed with HRP-streptavidin (blot #1). The blot was stripped and re-probed with anti-human RPN13 (blot #2). (B) Streptavidin bead-mediated pull down of RPN13 from rat IEC-6 cell lysate treated with biotinylated EF24 or CLEFMA. After blotting with anti-human HRP-streptavidin (blot #3), the membrane was stripped and re-probed with anti-rat RPN13 antibody (blot #4).

    Article Snippet: Anti-actin primary antibody, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody, and HRP-streptavidin were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Purification

    Interaction of EF24 and CLEFMA with proteasome. Highly purified (A,B) 26S proteasome, (C) 20S proteasome, and (D,E) 19S regulator were allowed to interact with biotinylated analogs of EF24 or CLEFMA (10 μM). The mixtures were separated on a regular 10% reducing gel and the transfer membranes were probed for biotin using HRP-streptavidin.

    Journal: Frontiers in Chemistry

    Article Title: Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

    doi: 10.3389/fchem.2018.00392

    Figure Lengend Snippet: Interaction of EF24 and CLEFMA with proteasome. Highly purified (A,B) 26S proteasome, (C) 20S proteasome, and (D,E) 19S regulator were allowed to interact with biotinylated analogs of EF24 or CLEFMA (10 μM). The mixtures were separated on a regular 10% reducing gel and the transfer membranes were probed for biotin using HRP-streptavidin.

    Article Snippet: Anti-actin primary antibody, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody, and HRP-streptavidin were from Sigma-Aldrich (St. Louis, MO).

    Techniques: Purification

    CD5 and CD6 ectodomains bind PSC tegumental antigens. ( A ) Biotin-labeled rshCD5, rshCD6 and BSA protein solutions (20 μg/mL) were sequentially incubated (4 times) with PSC suspensions (5,000 PSC; viability ≥90%), and pellets and solutions run on SDS-PAGE and further Western blotted with HRP-streptavidin. Lanes 1 and 2: protein solutions before (0x) and after 4 sequential incubations with PSC (4x), respectively. Lanes 3 to 7: PSC pellets after 0x to 4x sequential incubations. ( B ) ELISA assays showing the binding of increasing amounts of biotinylated rshCD5, rshCD6 and BSA proteins to PSEx-coated plates. ( C ) ELISA assays showing the binding of supernatants from sequential PSC incubations of biotin-labeled rshCD5, rshCD6 and BSA depicted in ( A ), to PSEx-coated plates. N.D. not detected. (*) Significant differences (Student’s t-test, P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis

    doi: 10.1371/journal.pntd.0006891

    Figure Lengend Snippet: CD5 and CD6 ectodomains bind PSC tegumental antigens. ( A ) Biotin-labeled rshCD5, rshCD6 and BSA protein solutions (20 μg/mL) were sequentially incubated (4 times) with PSC suspensions (5,000 PSC; viability ≥90%), and pellets and solutions run on SDS-PAGE and further Western blotted with HRP-streptavidin. Lanes 1 and 2: protein solutions before (0x) and after 4 sequential incubations with PSC (4x), respectively. Lanes 3 to 7: PSC pellets after 0x to 4x sequential incubations. ( B ) ELISA assays showing the binding of increasing amounts of biotinylated rshCD5, rshCD6 and BSA proteins to PSEx-coated plates. ( C ) ELISA assays showing the binding of supernatants from sequential PSC incubations of biotin-labeled rshCD5, rshCD6 and BSA depicted in ( A ), to PSEx-coated plates. N.D. not detected. (*) Significant differences (Student’s t-test, P

    Article Snippet: Bound protein was detected by the addition of 100 μL/well HRP-labelled streptavidin (1:5,000—Sigma) for 1 h at 37°C.

    Techniques: Labeling, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay

    Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with streptavidin-Cy3 (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using anti-His-HRP (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Topokaryotyping demonstrates single cell variability and stress dependent variations in nuclear envelope associated domains

    doi: 10.1093/nar/gky818

    Figure Lengend Snippet: Proximity Utilizing Biotinylation (PUB) allows for detection of nuclear envelope-proximal regions on mitotic chromosomes. ( A ) The Proximity Utilizing Biotinylation principle. The PUB approach is based on coexpression of protein X fused to biotin ligase (BirA) and protein Y fused to biotin acceptor peptide (BAP). When the proteins are in sufficient proximity or interact with each other in cells harboring the corresponding plasmids, in the presence of biotin (pink), BirA specifically biotinylates the BAP peptide, resulting in a persistent label on the BAP-carrying protein. ( B ) Emerin is localized at the NE. 293T cells were transfected with emerin-GFP (green) and stained with DAPI (blue). Scale bar: 10 μm. ( C ) Biotinylation of NE proximal chromatin using BirA-emerin fusion. 293T cells were transfected with BirA-emerin and BAP-H2A or BAP-H3.1, followed by biotin pulse labeling and counterstaining with streptavidin-Cy3 (red) and DAPI (blue). Scale bar: 10 μm. ( D ) The biotin label depends on BAP-histone biotinylation. Western blot analysis was performed on nuclei from control (untransfected) and transfected 293T cells using anti-His-HRP (left) or streptavidin-HRP (right). The bands corresponding to BAP-histones, biotinylated BAP-histones, the ubiquitinated form of BAP-H2A (Ub-BAP-H2A) and non-specific signals (NS) are indicated. ( E ) PUB pulse and chase principle. BirA is fused to a nuclear envelope (NE) protein and BAP to a histone resulting in biotin labeling of chromatin proximal to the NE during interphase. The resulting biotin signals appear as bands on mitotic chromosomes. ( F ) PUB pulse and chase experiment. Interphase 293T cells coexpressing BirA-emerin and BAP-H2A are biotin pulse labeled followed by streptavidin-Cy3 (red) and DAPI (blue) staining (top). After 4 h chase, biotin signal was revealed on mitotic chromosomes using the same staining dies as previously described (bottom). Scale bar: 10 μm.

    Article Snippet: Expression of the BAP-tagged proteins was detected with an HRP-conjugated anti-polyHistidine antibody at 1:4000 (Sigma A7058) and biotinylated proteins were visualized with an HRP-conjugated streptavidin at 1:1000 (Sigma S2438).

    Techniques: Transfection, Staining, Labeling, Western Blot

    Comparison of brevican, the ADAMTS-derived neoepitope of brevican and WFA reactivity in rat and mouse brain extracts . Western blot of brevican, EAV(M)ESE, and Wisteria floribunda agglutinin (WFA) in rodent brain extracts before and after chondroitinase digestion: Rat (Rt) and mouse (Ms) extracts were probed for (A) anti-brevican, (B) anti-EAVESE (Rt) or anti-EAMESE (Ms), or (C) biotinylated WFA: Samples were treated with (+) and without (-) Chondroitinase ABC (Chase). (A+) The 145 kD core protein of brevican increased after Chase treatment, (B+) the proteolytic brevican fragment remained unchanged, and (C+) only a high molecular weight WFA-reactive band was diminished in Ms. (C) After probing with WFA, multiple, unidentified lower molecular weight bands were observed along with less abundant, high molecular weight moieties. The right panel in (C) was probed with secondary, HRP-conjugated streptavidin alone, which revealed two, major non-specific bands. (D) After differential centrifugation of rat brain tissue, brevican immunoreactivity (left panel) was predominately found in the soluble fraction (S), whereas most of the WFA reactivity (right panel) was observed in the membrane

    Journal: BMC Neuroscience

    Article Title: Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain

    doi: 10.1186/1471-2202-9-14

    Figure Lengend Snippet: Comparison of brevican, the ADAMTS-derived neoepitope of brevican and WFA reactivity in rat and mouse brain extracts . Western blot of brevican, EAV(M)ESE, and Wisteria floribunda agglutinin (WFA) in rodent brain extracts before and after chondroitinase digestion: Rat (Rt) and mouse (Ms) extracts were probed for (A) anti-brevican, (B) anti-EAVESE (Rt) or anti-EAMESE (Ms), or (C) biotinylated WFA: Samples were treated with (+) and without (-) Chondroitinase ABC (Chase). (A+) The 145 kD core protein of brevican increased after Chase treatment, (B+) the proteolytic brevican fragment remained unchanged, and (C+) only a high molecular weight WFA-reactive band was diminished in Ms. (C) After probing with WFA, multiple, unidentified lower molecular weight bands were observed along with less abundant, high molecular weight moieties. The right panel in (C) was probed with secondary, HRP-conjugated streptavidin alone, which revealed two, major non-specific bands. (D) After differential centrifugation of rat brain tissue, brevican immunoreactivity (left panel) was predominately found in the soluble fraction (S), whereas most of the WFA reactivity (right panel) was observed in the membrane "insoluble" fraction (I) whereas anti-EAVESE immunoreactivity (middle panel) was evident in both fractions. (E) Rt and Ms samples were treated with Chase in the absence (-) and presence (+) of a protease inhibitor cocktail (left panel) and probed with biotinylated-WFA. The high molecular weight smear is eliminated after treatment with Chase, but the protease inhibitor did not change the pattern. (right panel) The same membrane was probed with anti-brevican where complete removal of CS chains led to an increase in abundance of the core protein with no change in the abundance of fragment. Protease inhibitor had no effect on Chase action.

    Article Snippet: Primary antibodies and biotinylated Wisteria floribunda lectin were detected with corresponding secondary antibodies including anti-mouse, anti-rabbit and streptavidin conjugated to horse radish peroxidase (Chemicon, Temecula, CA), respectively.

    Techniques: Derivative Assay, Western Blot, Mass Spectrometry, Molecular Weight, Centrifugation, Protease Inhibitor

    Cleavage of Sam68 by recombinant caspases. Cleavage of in vitro translated Sam68 by activated caspases. Biotin-labeled in vitro translated mouse Sam68 was incubated with 10 units of recombinant active caspase-1, -2, -3, -6, -7, -8, -9 and -10 for 1 h at 37°C. Caspase activity was blocked by pretreatment with caspase inhibitor, as indicated. The reactants were separated by SDS-PAGE and immunoblotted (IB) with streptavidin–HRP conjugate to detect full length or cleaved Sam68 proteins.

    Journal: Journal of Radiation Research

    Article Title: Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation

    doi: 10.1093/jrr/rru113

    Figure Lengend Snippet: Cleavage of Sam68 by recombinant caspases. Cleavage of in vitro translated Sam68 by activated caspases. Biotin-labeled in vitro translated mouse Sam68 was incubated with 10 units of recombinant active caspase-1, -2, -3, -6, -7, -8, -9 and -10 for 1 h at 37°C. Caspase activity was blocked by pretreatment with caspase inhibitor, as indicated. The reactants were separated by SDS-PAGE and immunoblotted (IB) with streptavidin–HRP conjugate to detect full length or cleaved Sam68 proteins.

    Article Snippet: Cleaved fragments were detected by anti-streptavidin-HRP (Sigma).

    Techniques: Recombinant, In Vitro, Labeling, Incubation, Activity Assay, SDS Page

    Purification of recombinant bovine Hsp70 and formation of Hsp70:peptide complexes. (A) Purified Hsp70 (1 μg) was resolved by SDS-PAGE on a 10% gel and detected by silver staining. Left hand lane indicates molecular weight markers. (B) Purified Hsp70 (250 ng) was analysed by Western blot using an anti-Hsp70 monoclonal antibody (SPA-810, Stressgen). (C) 2 μM Hsp70 was incubated with 60 μM biotinylated peptide and increasing amounts of unlabelled peptide in 55 μL PBS at 37 °C for 1 h to form complexes. Fractions containing an equivalent of 1 μg Hsp70 were resolved by non-reducing SDS-PAGE on a 10% gel, followed by Western blot analysis using streptavidin-HRP to detect biotinylated peptide. Controls of Hsp70 and biotinylated peptide only were run in lanes 1 and 2 respectively. Lanes 4–8 additionally contain unlabelled peptide at 6 μM, 60 μM, 150 μM, 300 μM and 600 μM (0.1×, 1×, 2.5×, 5× and 10× molar concentration of labelled peptide). (A color version of this figure is available at www.vetres.org .)

    Journal: Veterinary Research

    Article Title: Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

    doi: 10.1051/vetres/2010008

    Figure Lengend Snippet: Purification of recombinant bovine Hsp70 and formation of Hsp70:peptide complexes. (A) Purified Hsp70 (1 μg) was resolved by SDS-PAGE on a 10% gel and detected by silver staining. Left hand lane indicates molecular weight markers. (B) Purified Hsp70 (250 ng) was analysed by Western blot using an anti-Hsp70 monoclonal antibody (SPA-810, Stressgen). (C) 2 μM Hsp70 was incubated with 60 μM biotinylated peptide and increasing amounts of unlabelled peptide in 55 μL PBS at 37 °C for 1 h to form complexes. Fractions containing an equivalent of 1 μg Hsp70 were resolved by non-reducing SDS-PAGE on a 10% gel, followed by Western blot analysis using streptavidin-HRP to detect biotinylated peptide. Controls of Hsp70 and biotinylated peptide only were run in lanes 1 and 2 respectively. Lanes 4–8 additionally contain unlabelled peptide at 6 μM, 60 μM, 150 μM, 300 μM and 600 μM (0.1×, 1×, 2.5×, 5× and 10× molar concentration of labelled peptide). (A color version of this figure is available at www.vetres.org .)

    Article Snippet: An HRP-conjugated streptavidin antibody diluted 1/500 in blocking buffer was added for 45 min at room temperature before development with o-Phenylenediamine dihydrochloride (OPD; Sigma) according to manufacturer’s instructions.

    Techniques: Purification, Recombinant, SDS Page, Silver Staining, Molecular Weight, Western Blot, Incubation, Concentration Assay

    Biotinylated β 2 m-binding proteins from the surface of S. mansoni schistosomula. Bound proteins and unbound proteins were detected using HRP-streptavidin. Bound proteins are highlighted by arrows.

    Journal: Infection and Immunity

    Article Title: Receptor for Fc on the Surfaces of Schistosomes

    doi: 10.1128/IAI.69.6.3646-3651.2001

    Figure Lengend Snippet: Biotinylated β 2 m-binding proteins from the surface of S. mansoni schistosomula. Bound proteins and unbound proteins were detected using HRP-streptavidin. Bound proteins are highlighted by arrows.

    Article Snippet: Blots were probed with either horseradish peroxidase (HRP)-conjugated streptavidin (1 μg/ml; Sigma) or a rabbit antiserum (1:2,000) prepared against S. japonicum recombinant Pmy (r Sj -Pmy) ( ) for 1 h at room temperature (RT).

    Techniques: Binding Assay

    Co-immunoprecipitation assay (Co-IP). (A) Location of residue K265 in the 3D crystal structure of ZIKV NS1. (B) Flowchart of Co-IP experiments from ZIKV infected cells. Rabbit IgG anti-NS1 or control IgGs were used for pull down ZIKV NS1 and its associated complexes. (C) Co-IP results from (B). NS1 in both eluates and cell lysates were probed using rabbit anti-NS1 and protein A-HRP. E proteins were detected by mouse IgG anti-ZIKV and goat anti-mouse IgG-HRP. prM was examined by rabbit IgG anti-ZIKV prM and goat anti-rabbit IgG-HRP. (D) Densitometry analysis of Western blot results from (C). The band intensities of prM and E proteins in (C) were quantified and normalized to those of NS1 proteins from corresponding eluates. The efficiencies of prM and E pulled-down by WT NS1 were set as 100%. The averaged relative intensities from three independent experiments were shown. An unpaired Student t- test was used to estimate the statistical significance. (E) Flowchart of Co-IP from HEK293T cells transiently expressing NS1 and NS2A-HA. Cell lysates was subjected to Co-IP using mouse IgG anti-HA or mouse control IgG. (F) Western blot results from (E). NS2A-HA in the eluates and cell lysates were examined by rabbit IgG anti-HA and goat anti-rabbit IgG-HRP respectively. NS1 proteins were detected by rabbit anti-ZIKV NS1 and goat anti-rabbit IgG-HRP. (G) Densitometry analysis of Western blot results from (F). The band intensities of NS1 proteins in (F) were quantified and normalized to those of NS2A-HA proteins from corresponding eluates. An unpaired Student t- test was used to evaluate the statistical significance.

    Journal: EBioMedicine

    Article Title: A cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine

    doi: 10.1016/j.ebiom.2017.02.003

    Figure Lengend Snippet: Co-immunoprecipitation assay (Co-IP). (A) Location of residue K265 in the 3D crystal structure of ZIKV NS1. (B) Flowchart of Co-IP experiments from ZIKV infected cells. Rabbit IgG anti-NS1 or control IgGs were used for pull down ZIKV NS1 and its associated complexes. (C) Co-IP results from (B). NS1 in both eluates and cell lysates were probed using rabbit anti-NS1 and protein A-HRP. E proteins were detected by mouse IgG anti-ZIKV and goat anti-mouse IgG-HRP. prM was examined by rabbit IgG anti-ZIKV prM and goat anti-rabbit IgG-HRP. (D) Densitometry analysis of Western blot results from (C). The band intensities of prM and E proteins in (C) were quantified and normalized to those of NS1 proteins from corresponding eluates. The efficiencies of prM and E pulled-down by WT NS1 were set as 100%. The averaged relative intensities from three independent experiments were shown. An unpaired Student t- test was used to estimate the statistical significance. (E) Flowchart of Co-IP from HEK293T cells transiently expressing NS1 and NS2A-HA. Cell lysates was subjected to Co-IP using mouse IgG anti-HA or mouse control IgG. (F) Western blot results from (E). NS2A-HA in the eluates and cell lysates were examined by rabbit IgG anti-HA and goat anti-rabbit IgG-HRP respectively. NS1 proteins were detected by rabbit anti-ZIKV NS1 and goat anti-rabbit IgG-HRP. (G) Densitometry analysis of Western blot results from (F). The band intensities of NS1 proteins in (F) were quantified and normalized to those of NS2A-HA proteins from corresponding eluates. An unpaired Student t- test was used to evaluate the statistical significance.

    Article Snippet: The following antibodies were used: a mouse monoclonal antibody (mAb) 4G2 cross-reactive with flavivirus E protein (ATCC); goat anti-mouse IgG conjugated with Alexa Fluor®488 (Thermo Fisher Scientific, Waltham, MA); goat anti-mouse or anti-rabbit IgGs conjugated with horseradish peroxidase (IgG-HRP), protein A conjugated with HRP (A-HRP; Sigma, St. Louis, MO); rabbit or mouse control IgGs (ThermoFisher Scientific); rabbit IgG against ZIKV NS1, mouse IgG anti-ZIKV E, rabbit IgG anti-ZIKV prM (Alpha Diagnostic Intl.

    Techniques: Co-Immunoprecipitation Assay, Infection, Western Blot, Expressing

    Determination of GP1/PS ratios in rLCMV-LASVGP and authentic LASV. (A) Schematic of the capture ELISA. Virus is captured by immobilized MAb 86.3, which recognizes a conserved nonneutralizing epitope in GP2. After removal of unbound material, bound virus is probed with ANX-V to detect PS, displayed in the lipid bilayer of the viral envelope, and DGEKFc4, which binds to GP1. For details, please see the text. (B) Detection of GP1 and PS. Purified rLCMV-LASVGP and authentic inactivated LASV were incubated with immobilized MAb 83.6, with the nonenveloped AdV5-GFP used as a negative control. After the plates were washed, bound virus was incubated with biotin-conjugated ANX-V in the presence and absence of EGTA, as well as DGEKFc4. Bound biotinylated ANX-V and DGEKFc4 were detected by streptavidin-HRP- and HRP-conjugated anti-human Fc antibody in a color reaction. Please note the reduced ANX-V binding in the presence of the Ca 2+ chelator EGTA. Data are means ± SD ( n = 3). (C) Ratios of GP1 to PS calculated for the virus preparations tested in panel B. OD (450), optical density at 450 nm; LASVi, inactivated LASV.

    Journal: Journal of Virology

    Article Title: Axl Can Serve as Entry Factor for Lassa Virus Depending on the Functional Glycosylation of Dystroglycan

    doi: 10.1128/JVI.01613-17

    Figure Lengend Snippet: Determination of GP1/PS ratios in rLCMV-LASVGP and authentic LASV. (A) Schematic of the capture ELISA. Virus is captured by immobilized MAb 86.3, which recognizes a conserved nonneutralizing epitope in GP2. After removal of unbound material, bound virus is probed with ANX-V to detect PS, displayed in the lipid bilayer of the viral envelope, and DGEKFc4, which binds to GP1. For details, please see the text. (B) Detection of GP1 and PS. Purified rLCMV-LASVGP and authentic inactivated LASV were incubated with immobilized MAb 83.6, with the nonenveloped AdV5-GFP used as a negative control. After the plates were washed, bound virus was incubated with biotin-conjugated ANX-V in the presence and absence of EGTA, as well as DGEKFc4. Bound biotinylated ANX-V and DGEKFc4 were detected by streptavidin-HRP- and HRP-conjugated anti-human Fc antibody in a color reaction. Please note the reduced ANX-V binding in the presence of the Ca 2+ chelator EGTA. Data are means ± SD ( n = 3). (C) Ratios of GP1 to PS calculated for the virus preparations tested in panel B. OD (450), optical density at 450 nm; LASVi, inactivated LASV.

    Article Snippet: Annexin V from human placenta conjugated to biotin, heparin, and streptavidin-HRP were from Sigma.

    Techniques: Enzyme-linked Immunosorbent Assay, Purification, Incubation, Negative Control, Binding Assay

    ). As a negative control for Axl, HEK293H cells were included. The negative-control lane of the Axl blot was taken from the same membrane and moved, as indicated by the thin white line. Primary antibodies were detected with HRP-conjugated secondary antibodies using enhanced chemiluminescence (ECL) for development. The observed differences in the apparent molecular masses of Axl in different cells were consistently observed and may be due to different glycosylation patterns. The expression levels of Axl in PHHC varied between donors, and a representative example was selected. (B) Western blot of WGA-purified DG. Dystroglycan was purified from the indicated cells by WGA affinity chromatography. Concentrated fractions were probed in a Western blot for functional glycosylation of α-DG with MAb IIH6 and for β-DG with MAb 8D5 as described for panel A. (C) Solid-phase virus binding assay. Equal amounts of DG purified from the indicated cells were immobilized in microtiter plates and incubated with the indicated concentrations of purified rLCMV-LASVGP. Bound virus was detected with MAb 83.6 to LASV GP2 using a biotinylated secondary antibody and HRP-conjugated streptavidin in a color reaction. Data are means ± standard deviations (SD) ( n = 3).

    Journal: Journal of Virology

    Article Title: Axl Can Serve as Entry Factor for Lassa Virus Depending on the Functional Glycosylation of Dystroglycan

    doi: 10.1128/JVI.01613-17

    Figure Lengend Snippet: ). As a negative control for Axl, HEK293H cells were included. The negative-control lane of the Axl blot was taken from the same membrane and moved, as indicated by the thin white line. Primary antibodies were detected with HRP-conjugated secondary antibodies using enhanced chemiluminescence (ECL) for development. The observed differences in the apparent molecular masses of Axl in different cells were consistently observed and may be due to different glycosylation patterns. The expression levels of Axl in PHHC varied between donors, and a representative example was selected. (B) Western blot of WGA-purified DG. Dystroglycan was purified from the indicated cells by WGA affinity chromatography. Concentrated fractions were probed in a Western blot for functional glycosylation of α-DG with MAb IIH6 and for β-DG with MAb 8D5 as described for panel A. (C) Solid-phase virus binding assay. Equal amounts of DG purified from the indicated cells were immobilized in microtiter plates and incubated with the indicated concentrations of purified rLCMV-LASVGP. Bound virus was detected with MAb 83.6 to LASV GP2 using a biotinylated secondary antibody and HRP-conjugated streptavidin in a color reaction. Data are means ± standard deviations (SD) ( n = 3).

    Article Snippet: Annexin V from human placenta conjugated to biotin, heparin, and streptavidin-HRP were from Sigma.

    Techniques: Negative Control, Expressing, Western Blot, Whole Genome Amplification, Purification, Affinity Chromatography, Functional Assay, Binding Assay, Incubation

    Biochemical profiling of the F42 (SRVWKAPQYSDEFV) peptide ligand. (A) Alanine scanning mutagenesis. (B) N-terminal deletion analysis. (C) C-terminal deletion analysis. The sequences indicated were synthesized as biotinylated peptides, conjugated to streptavidin-HRP

    Journal:

    Article Title: An unconventional IAP-binding motif revealed by target-assisted iterative screening (TAIS) of the BIR3-cIAP1 domain

    doi: 10.1002/jmr.809

    Figure Lengend Snippet: Biochemical profiling of the F42 (SRVWKAPQYSDEFV) peptide ligand. (A) Alanine scanning mutagenesis. (B) N-terminal deletion analysis. (C) C-terminal deletion analysis. The sequences indicated were synthesized as biotinylated peptides, conjugated to streptavidin-HRP

    Article Snippet: Individual biotinylated peptides (30 ng) were pre-incubated with 1 μg of streptavidin-HRP conjugate (Sigma) in 300 μl of TBS-T for 30 min at RT.

    Techniques: Mutagenesis, Synthesized

    NFATC3 selectively binds to IRF7 and enhances its transcriptional activity. (A) Fresh isolated human primary pDCs or pDCs stimulated with CpG A for 4 h. Cells were fixed with paraformaldehyde, permeablized, stained with anti-IRF7 (green) and anti-NFATC3 (red), and mounted with Prolong gold antifade reagent with DAPI (blue). Cells were analyzed using confocal microscopy. (B) Untouched human pDCs were isolated as described in the Confocal microscopy section of Materials and methods. pDCs were left untreated or treated with 1 µM CpG A for 3 h, and pDCs were lysed and immunoprecipitated (IP) with NFATC3 antibody. IRF7 was detected by immunoblotting (IB). (C) Purified GST or GST-tagged NFATC3 were incubated with Myc-tagged IRF7. Myc-tagged IRF7 was immunoprecipitated with Myc–agarose beads. GST was detected by anti-GST HRP conjugate antibody. (D) Purified Myc-tagged IRF3 or IRF7 was incubated with GST-tagged NFATC3, and GST-tagged NFATC3 was immunoprecipitated with glutathione Sepharose beads. Then, Myc-tagged proteins were detected by anti-Myc antibody. (E) HEK-293T cells were transfected with HA-tagged NFAT plasmids and Myc-tagged IRF7; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (F) HEK-293T cells were transfected with HA-tagged NFATC3 plasmids and Myc-tagged MyD88, IRF3, and IRF7 plasmids; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (G and H) HEK-293T cells were transfected with 100 ng IFN-α4 luciferase reporter plasmid (IFNα4-Luc) or IFN-β luciferase reporter plasmid (IFNβ-Luc) and 0.5 ng IRF7 expression vector together with an increasing amount of NFATC3 expression vector (25, 50, and 100 ng). 0.1 ng renilla luciferase reporter plasmid was transfected simultaneously as an internal control. Results are presented as fold induction relative to the activity of renilla luciferase. (I) HEK-293T cells were transfected with 100 ng NF-κB luciferase reporter plasmid (NFκB-Luc) together with 5 ng MyD88 expression vector or an increasing amount of NFATC3 expression vector. (J) HEK-293T cells were transfected with 100 ng IFN-β luciferase reporter plasmid (IFNβ-Luc) together with 5 ng IKKi expression vector or an increasing amount of NFATC3 expression vector. (G–J) Data are representative of four independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

    doi: 10.1084/jem.20160438

    Figure Lengend Snippet: NFATC3 selectively binds to IRF7 and enhances its transcriptional activity. (A) Fresh isolated human primary pDCs or pDCs stimulated with CpG A for 4 h. Cells were fixed with paraformaldehyde, permeablized, stained with anti-IRF7 (green) and anti-NFATC3 (red), and mounted with Prolong gold antifade reagent with DAPI (blue). Cells were analyzed using confocal microscopy. (B) Untouched human pDCs were isolated as described in the Confocal microscopy section of Materials and methods. pDCs were left untreated or treated with 1 µM CpG A for 3 h, and pDCs were lysed and immunoprecipitated (IP) with NFATC3 antibody. IRF7 was detected by immunoblotting (IB). (C) Purified GST or GST-tagged NFATC3 were incubated with Myc-tagged IRF7. Myc-tagged IRF7 was immunoprecipitated with Myc–agarose beads. GST was detected by anti-GST HRP conjugate antibody. (D) Purified Myc-tagged IRF3 or IRF7 was incubated with GST-tagged NFATC3, and GST-tagged NFATC3 was immunoprecipitated with glutathione Sepharose beads. Then, Myc-tagged proteins were detected by anti-Myc antibody. (E) HEK-293T cells were transfected with HA-tagged NFAT plasmids and Myc-tagged IRF7; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (F) HEK-293T cells were transfected with HA-tagged NFATC3 plasmids and Myc-tagged MyD88, IRF3, and IRF7 plasmids; 36 h after transfection, cell lysates were immunoprecipitated with anti-HA beads, and then the immunoprecipitates and lysates were analyzed with anti-HA or anti-Myc antibodies. (G and H) HEK-293T cells were transfected with 100 ng IFN-α4 luciferase reporter plasmid (IFNα4-Luc) or IFN-β luciferase reporter plasmid (IFNβ-Luc) and 0.5 ng IRF7 expression vector together with an increasing amount of NFATC3 expression vector (25, 50, and 100 ng). 0.1 ng renilla luciferase reporter plasmid was transfected simultaneously as an internal control. Results are presented as fold induction relative to the activity of renilla luciferase. (I) HEK-293T cells were transfected with 100 ng NF-κB luciferase reporter plasmid (NFκB-Luc) together with 5 ng MyD88 expression vector or an increasing amount of NFATC3 expression vector. (J) HEK-293T cells were transfected with 100 ng IFN-β luciferase reporter plasmid (IFNβ-Luc) together with 5 ng IKKi expression vector or an increasing amount of NFATC3 expression vector. (G–J) Data are representative of four independent experiments.

    Article Snippet: The following antibodies were used for immunoblot analysis and/or immunoprecipitation: anti-IRF7 (sc-74472), anti-IRF7 (sc-9083), anti-NFATC1 (sc-13033), anti-NFATC2 (sc-7296), anti-NFATC3 (sc-8321), anti-NFATC4 (sc-13036), and anti-NFAT5 (sc-13035) were from Santa Cruz Biotechnology, Inc.; anti-IRF7 (51–3300) was from Thermo Fisher Scientific; anti-MyD88 (4283), anti–NF-κB1 p105/p50, and anti-HDAC1 (5356) were from Cell Signaling Technology; and anti-NFATC3 (A303-572A) was from Bethyl Laboratories, Inc. HRP-conjugated anti-HA (ab1265), anti-Myc (ab62928), and anti-transferrin receptor antibody (ab84036) were from Abcam; anti–β-actin, anti-HA antibodies, anti-GST HRP conjugate antibody, HA–agarose beads, and Myc–agarose beads were purchased from Sigma-Aldrich.

    Techniques: Activity Assay, Isolation, Staining, Confocal Microscopy, Immunoprecipitation, Purification, Incubation, Transfection, Luciferase, Plasmid Preparation, Expressing

    The specific interactions between Cry6Aa and ASP-1. (A) The expression and purification of ASP1-GST fusion protein in E . coli . Lane 1, E . coli BL21 containing pGEX-6p-1 vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet; lane 2, E . coli BL21 containing pASP1-GSTvector without IPTG induction, cell pellet. lane 3, E . coli BL21 containing pASP1-GST vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet. lane 4, purified ASP1-GST fusion protein eluted from the glutathione Sepharose bulk; lane 5, purified GST tag eluted from the glutathione Sepharose bulk. (B) The binding of Cry6Aa to ASP1-GST fusion proteins by ligand blotting. The purified ASP1-GST fusion proteins (lane 6) or GST (lane 7) were separated by SDS-PAGE gels, and were transferred to a PVDF membrane. Filters were blocked overnight, and then probed with biotinylated Cry6Aa, Unbound toxin was removed by washing. The bound protein was detected streptavidin-horseradish peroxidase (HRP) conjugate. And finally the membrane was visualized. GST was the control. (C) ASP-1 proteins were dotted on a NC membrane directly and were probed with biotin labeled Cry6Aa or with biotin labeled Cry6Aa plus unlabeled Cry6Aa (1000-fold). GST was the control. (D) Binding affinity of Cry6Aa to ASP-1 was determined by ELISA. Ninety-six-well microtiter plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry6Aa alone or with 1000-fold molar excess of unlabeled Cry6Aa to determine specific binding. Each point represents mean amounts of protein specifically bound. Specific binding was determined by subtracting nonspecific binding (with 1000-fold molar excess of unlabeled Cry6Aa) from total binding (without excess unlabeled Cry6Aa). (E) Isothermal titration calorimetric analysis of Cry6Aa binding to ASP-1. Titration of ASP-1 (2.20 μM) with 21.16 μM Cry6Aa. The top panel show the raw data of the heat released, and the bottom panel show the binding isotherm fitted using nonlinear binding models. Data were analyzed in Origin 8.6 software after subtracting the heat released from titrating Cry6Aa alone into buffer. One of three representative experiments is shown.

    Journal: PLoS Pathogens

    Article Title: Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    doi: 10.1371/journal.ppat.1005389

    Figure Lengend Snippet: The specific interactions between Cry6Aa and ASP-1. (A) The expression and purification of ASP1-GST fusion protein in E . coli . Lane 1, E . coli BL21 containing pGEX-6p-1 vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet; lane 2, E . coli BL21 containing pASP1-GSTvector without IPTG induction, cell pellet. lane 3, E . coli BL21 containing pASP1-GST vector with 0.1 mM IPTG induction under 16°C overnight, cell pellet. lane 4, purified ASP1-GST fusion protein eluted from the glutathione Sepharose bulk; lane 5, purified GST tag eluted from the glutathione Sepharose bulk. (B) The binding of Cry6Aa to ASP1-GST fusion proteins by ligand blotting. The purified ASP1-GST fusion proteins (lane 6) or GST (lane 7) were separated by SDS-PAGE gels, and were transferred to a PVDF membrane. Filters were blocked overnight, and then probed with biotinylated Cry6Aa, Unbound toxin was removed by washing. The bound protein was detected streptavidin-horseradish peroxidase (HRP) conjugate. And finally the membrane was visualized. GST was the control. (C) ASP-1 proteins were dotted on a NC membrane directly and were probed with biotin labeled Cry6Aa or with biotin labeled Cry6Aa plus unlabeled Cry6Aa (1000-fold). GST was the control. (D) Binding affinity of Cry6Aa to ASP-1 was determined by ELISA. Ninety-six-well microtiter plates coated with ASP-1 were incubated with increasing concentrations of biotinylated Cry6Aa alone or with 1000-fold molar excess of unlabeled Cry6Aa to determine specific binding. Each point represents mean amounts of protein specifically bound. Specific binding was determined by subtracting nonspecific binding (with 1000-fold molar excess of unlabeled Cry6Aa) from total binding (without excess unlabeled Cry6Aa). (E) Isothermal titration calorimetric analysis of Cry6Aa binding to ASP-1. Titration of ASP-1 (2.20 μM) with 21.16 μM Cry6Aa. The top panel show the raw data of the heat released, and the bottom panel show the binding isotherm fitted using nonlinear binding models. Data were analyzed in Origin 8.6 software after subtracting the heat released from titrating Cry6Aa alone into buffer. One of three representative experiments is shown.

    Article Snippet: The bound protein was detected by 1 μg/ml streptavidin- horseradish peroxidase (HRP) conjugate (Sigma, S5512).

    Techniques: Expressing, Purification, Plasmid Preparation, Binding Assay, SDS Page, Labeling, Enzyme-linked Immunosorbent Assay, Incubation, Titration, Software

    Affinity precipitation (A) and blot overlay (B) analysis of the interaction between palladin and ezrin. (A) Yeast lysates expressing HA-tagged palladin Ig1, Ig2–3, and ezrin 1–309 were incubated with glutathione-Sepharose–coupled GST-ezrin 279–531, GST-ezrin 477–585, and GST. After washes bound material was eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters. Proteins were detected by HA-immunoblotting with the use of 12CA5 mAb. Palladin Ig2–3 interacts with the α-helical domain of ezrin (279–531) but not with the C-terminal domain (477–585). As a positive control, the association of ezrin 1–309 and ezrin 477–585 is shown. (B) Total lysates of HISM and U251mg cells, purified ezrin, or recombinant ezrin 1–309 were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with biotinylated probes GST, GST-palladin Ig2–3, and GST-palladin Ig1–3. Bound probe was detected with the use of HRP-conjugated extravidin and enhanced chemiluminiscence. Both palladin probes bind to two (HISM) or three (U251 mg) major protein bands. Reprobing of the blots with ezrin antibody after stripping shows that the 75-kDa band recognized by palladin is consistent with being ezrin. The probes also react with immobilized purified ezrin but not with the N-terminal domain.

    Journal: Molecular Biology of the Cell

    Article Title: Characterization of Human Palladin, a Microfilament-associated Protein

    doi:

    Figure Lengend Snippet: Affinity precipitation (A) and blot overlay (B) analysis of the interaction between palladin and ezrin. (A) Yeast lysates expressing HA-tagged palladin Ig1, Ig2–3, and ezrin 1–309 were incubated with glutathione-Sepharose–coupled GST-ezrin 279–531, GST-ezrin 477–585, and GST. After washes bound material was eluted by boiling in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters. Proteins were detected by HA-immunoblotting with the use of 12CA5 mAb. Palladin Ig2–3 interacts with the α-helical domain of ezrin (279–531) but not with the C-terminal domain (477–585). As a positive control, the association of ezrin 1–309 and ezrin 477–585 is shown. (B) Total lysates of HISM and U251mg cells, purified ezrin, or recombinant ezrin 1–309 were run in SDS-PAGE and transferred to nitrocellulose filters. The filters were incubated with biotinylated probes GST, GST-palladin Ig2–3, and GST-palladin Ig1–3. Bound probe was detected with the use of HRP-conjugated extravidin and enhanced chemiluminiscence. Both palladin probes bind to two (HISM) or three (U251 mg) major protein bands. Reprobing of the blots with ezrin antibody after stripping shows that the 75-kDa band recognized by palladin is consistent with being ezrin. The probes also react with immobilized purified ezrin but not with the N-terminal domain.

    Article Snippet: The blots were incubated with the biotinylated probe, GST, GST-palladin Ig2–3, and GST-palladin Ig1–3 (0.1 μg/ml) in 1% bovine serum albumin in Tris-buffered saline-0.1% Tween 20 for 2 h. HRP-conjugated extravidin (Sigma) (dilution 1:10.000) was detected with the use of enhanced chemiluminescence.

    Techniques: Affinity Precipitation, Expressing, Incubation, SDS Page, Positive Control, Purification, Recombinant, Stripping Membranes

    A27 viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat anti-rabbit-HRP antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p

    Journal: Viruses

    Article Title: Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    doi: 10.3390/v5030928

    Figure Lengend Snippet: A27 viral protein interacts with SP-D. (A) Characterization of the recombinant A27 protein. The purified recombinant A27 protein (0.5 µg/lane and 2 µg/lane for Western-blot (left panel) and 12% SDS-PAGE analysis (right panel), respectively) was heated at 100°C for 5 min in denaturating, reducing sample buffer (lane 1) or incubated for 5 min at room temperature in non-reducing sample buffer (lane 2). RhSP-D (2.5 µg/ml) (B) or hNCRD and mutNCRD (5 µg/ml) (C) were incubated for 1 hour at 37°C with coated proteins on 96-multiwell plate. Bound RhSP-D was revealed with a rabbit anti-SP-D specific antibody and with a goat anti-rabbit-HRP antibody. TMB substrate was added and OD was measured at 450 nm. An asterisk indicates a statistically significant difference (n=3; p

    Article Snippet: ELISA assay for measurement of binding of NCRDs to A27 recombinant VACV protein Binding of trimeric NCRD fusion proteins to A27 protein of VACV was measured as previously described using the S protein-HRP conjugate (Novagen) [ ].

    Techniques: Recombinant, Purification, Western Blot, SDS Page, Incubation

    Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with HRP-conjugated avidin. C: control. 1–7: patients 1–7. The α subunit of 3-methylcrotonyl-CoA carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls

    Journal: JIMD Reports

    Article Title: Novel Mutations in the PC Gene in Patients with Type B Pyruvate Carboxylase Deficiency

    doi: 10.1007/8904_2012_173

    Figure Lengend Snippet: Analysis of PC protein and sequence analysis. ( a ) cDNA analysis of an exon 8–14 fragment. The normal size fragment of 829 bp is seen in the control (C), whereas a fragment of 684 bp with skipping of exon 12 is seen in patients 1 and 7. Patient 1 also has a band of 836 bp, which corresponds to a fragment with retention of the first 7 nucleotides of intron 8, caused by a splice site mutation, c.903+1G > A. The band of 897 bp in patient 7 represents a fragment with retention of the last 68 bp of intron 11. The intensity of the cDNA fragments in patients 1 and 7 is likely a consequence of activated NMD pathway. ( b ) Alignment of PC showing the conservation of the missense mutations found in patients 5 (p.Arg205Ser) and 6 (p.Gly869Asp). ( c ) The genomic sequence of exon 12 and the flanking ~40 bp of intron 11. The c.1369-29A > G mutation (in bold ) is located in a potential branch point sequence ( boxed ). ( d ) Western blot analysis of protein from fibroblast mitochondria with HRP-conjugated avidin. C: control. 1–7: patients 1–7. The α subunit of 3-methylcrotonyl-CoA carboxylase (3-MCC) and the α subunit of propionyl-CoA carboxylase were used as loading controls

    Article Snippet: Mitochondrial biotin–containing proteins (pyruvate carboxylase, the α subunit of 3-methylcrotonyl-CoA carboxylase and the α subunit of propionyl-CoA carboxylase) were detected with HRP-conjugated avidin (Sigma) at a 1:10,000 dilution and Supersignal West Pico substrate (Pierce) ( ).

    Techniques: Sequencing, Mutagenesis, Western Blot, Avidin-Biotin Assay

    Detection of in vitro expressed and biotinylated products. Four constructs were investigated for their ability to bind to neutravidin-coated microwells after in vitro expression and biotinylation: β-lactamase (amp), chloramphenicol acetyltransferase (cat), aminoglycoside phosphotransferase (neo) and maltose-binding protein (mbp). Purified, translated and biotinylated products were serially diluted and probed with their respective antisera and again with anti-rabbit IgG–HRP. Signal intensity is measured after incubation of the bound complexes with o -phenylenediamine at 492 nm.

    Journal: Nucleic Acids Research

    Article Title: Automated selection of aptamers against protein targets translated in vitro: from gene to aptamer

    doi:

    Figure Lengend Snippet: Detection of in vitro expressed and biotinylated products. Four constructs were investigated for their ability to bind to neutravidin-coated microwells after in vitro expression and biotinylation: β-lactamase (amp), chloramphenicol acetyltransferase (cat), aminoglycoside phosphotransferase (neo) and maltose-binding protein (mbp). Purified, translated and biotinylated products were serially diluted and probed with their respective antisera and again with anti-rabbit IgG–HRP. Signal intensity is measured after incubation of the bound complexes with o -phenylenediamine at 492 nm.

    Article Snippet: The other membrane was probed with a 1:100 000 dilution of avidin-conjugated HRP (Sigma-Aldrich, St Louis, MO) to detect biotinylated products.

    Techniques: In Vitro, Construct, Expressing, Binding Assay, Purification, Incubation

    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of EXOG-FLAG-tagged was monitored by Western analysis with FLAG-HRP conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.

    Journal: PLoS ONE

    Article Title: Deficiency in Repair of the Mitochondrial Genome Sensitizes Proliferating Myoblasts to Oxidative Damage

    doi: 10.1371/journal.pone.0075201

    Figure Lengend Snippet: Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of EXOG-FLAG-tagged was monitored by Western analysis with FLAG-HRP conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.

    Article Snippet: Western analysis was performed as described earlier [ ] with the membranes sequentially probed using antibodies against: DNA Ligase 3 (QED Bioscience Inc., San Diego, CA), Pax7 (R & D Systems, Minneapolis, MN), PCNA and myogenin (Santa Cruz Biotechnology, Santa Cruz, CA), nucleolin (Abcam, Cambridge, UK), APE1 (Novus Biologicals, Littleton, CO), GAPDH, caspase-9, caspase-8 and caspase-3 (Cell Signaling Technology, Danvers, MA), the 56KDa subunit of ATP synthase (Molecular Probe-Invitrogen, Carlsbad, CA) and FLAG-HRP conjugated (SIGMA, St. Louis, MO).

    Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Isolation

    MAGE proteins interact with RING-containing proteins. ( A ) Summary of the MAGE-RING interaction results. MAGEA1(1–309) (A1), MAGEB1(63–315) (B1), MAGEC2(129–339) (C2), MAGEE2(87–505) (E2), MAGEF1(74–260) (F1) and/or MAGEL2(420–630) (L2) baits were used to screen Y2H library of RING-finger preys. The Y2H interactions are shown: black - high confidence Y2H interactions observed on both -His and -Ade selection; gray - low confidence interactions (from Table S1 ). Then, selected MAGE-RING interactions were tested using co-immunoprecipitation and pull-down assays: +++ means strong interaction; ++ means intermediate strength of interaction; + means weak interaction; - means no interaction (based on results shown in Figs. S1 , S2 and S4 ); ND – not determined. Nine out of 14 MAGE-RING interactions detected in Y2H were confirmed. ( B ) Two examples of the MAGE-RING co-immunoprecipitation results. His-S-tag-MAGEA1 (lanes 1–3 and 7–9) was co-transfected with either GFP-TRIM8 (lanes 1–6) or myc-TRIM31 (lanes 7–12) into HEK293T cells. The HEK293T cell extracts were incubated with protein-S-beads and immunoprecipitated proteins were analyzed on western blots. MAGEA1 was detected using protein-S-HRP conjugate and the RING-finger-containing proteins were visualized using anti-GFP and/or anti-myc antibody, respectively. In control experiments, the pTriEx4 vector was co-transfected with either GFP-TRIM8 (lanes 4–6) or myc-TRIM31 (lanes 10–12). The input (I, 1% of lysate), unbound (U, 1% of lysate) and bound (B, 10% of lysate) fractions were separated by 12% SDS-PAGE.

    Journal: Cell Cycle

    Article Title: The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex

    doi: 10.1080/15384101.2014.1000112

    Figure Lengend Snippet: MAGE proteins interact with RING-containing proteins. ( A ) Summary of the MAGE-RING interaction results. MAGEA1(1–309) (A1), MAGEB1(63–315) (B1), MAGEC2(129–339) (C2), MAGEE2(87–505) (E2), MAGEF1(74–260) (F1) and/or MAGEL2(420–630) (L2) baits were used to screen Y2H library of RING-finger preys. The Y2H interactions are shown: black - high confidence Y2H interactions observed on both -His and -Ade selection; gray - low confidence interactions (from Table S1 ). Then, selected MAGE-RING interactions were tested using co-immunoprecipitation and pull-down assays: +++ means strong interaction; ++ means intermediate strength of interaction; + means weak interaction; - means no interaction (based on results shown in Figs. S1 , S2 and S4 ); ND – not determined. Nine out of 14 MAGE-RING interactions detected in Y2H were confirmed. ( B ) Two examples of the MAGE-RING co-immunoprecipitation results. His-S-tag-MAGEA1 (lanes 1–3 and 7–9) was co-transfected with either GFP-TRIM8 (lanes 1–6) or myc-TRIM31 (lanes 7–12) into HEK293T cells. The HEK293T cell extracts were incubated with protein-S-beads and immunoprecipitated proteins were analyzed on western blots. MAGEA1 was detected using protein-S-HRP conjugate and the RING-finger-containing proteins were visualized using anti-GFP and/or anti-myc antibody, respectively. In control experiments, the pTriEx4 vector was co-transfected with either GFP-TRIM8 (lanes 4–6) or myc-TRIM31 (lanes 10–12). The input (I, 1% of lysate), unbound (U, 1% of lysate) and bound (B, 10% of lysate) fractions were separated by 12% SDS-PAGE.

    Article Snippet: The amount of S-tagged proteins was analyzed on western blots using protein-S-HRP conjugate (Merck Millipore, http://www.emdmillipore.com/life-science-research/s-protein-hrp-conjugate/EMD_BIO-69047 ).

    Techniques: Selection, Immunoprecipitation, Transfection, Incubation, Western Blot, Plasmid Preparation, SDS Page

    TRIM31 can form TRIM31-MAGEA1-NSE4 complexes reminiscent of NSE1-NSE3-NSE4 trimers. ( A ) myc-FLAG-TRIM31 (top panel, lanes 1–6), MAGEA1 (middle panel, lanes 1–3 and 7–9) and His-S-tag-NSE4a (bottom panel, lanes 1–9) constructs were co-transfected into HEK293T cells. The cell extracts were precipitated using the anti-FLAG-beads and immunoprecipitated proteins were analyzed on western blots. TRIM31 was detected using anti-myc, NSE4a was visualized using protein-S-HRP conjugate and the MAGEA1 protein was detected using anti-MAGEA1 antibody. Note that the endogenous levels of MAGEA1 in HEK293T cells are very low (lanes 4–6) compared to the levels of ectopically expressed MAGEA1 (lanes 1–3). These data suggest that TRIM31 binds directly to NSE4a and forms TRIM31-MAGEA1-NSE4a complex. ( B ) Cartoon comparing common features of the NSE1-NSE3-NSE4 and the TRIM31-MAGEA1-NSE4 complexes. The NSE3/MAGE proteins share conserved hydrophobic pockets (within the WH/B subdomain) interacting with NSE4/EID proteins. NSE3/MAGEG1 (WH/A) binds to NSE1 and stimulates (arrow) its RING-finger (RING) E3 ubiquitin-ligase activity. Similarly, MAGEA1 binds to TRIM31 and stimulates (arrow) its RING-finger (RING) E3 ubiquitin-ligase activity. Both, NSE3/MAGEG1 and MAGEA1, require their WH/A binding motifs for their stimulatory effects (arrowheads). As the TRIM31-MAGEA1-NSE4 complex shares common features with the yeast and human NSE1-NSE3-NSE4 complexes we propose that they may have evolved from a common ancestral NSE1-NSE3-NSE4 complex.

    Journal: Cell Cycle

    Article Title: The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex

    doi: 10.1080/15384101.2014.1000112

    Figure Lengend Snippet: TRIM31 can form TRIM31-MAGEA1-NSE4 complexes reminiscent of NSE1-NSE3-NSE4 trimers. ( A ) myc-FLAG-TRIM31 (top panel, lanes 1–6), MAGEA1 (middle panel, lanes 1–3 and 7–9) and His-S-tag-NSE4a (bottom panel, lanes 1–9) constructs were co-transfected into HEK293T cells. The cell extracts were precipitated using the anti-FLAG-beads and immunoprecipitated proteins were analyzed on western blots. TRIM31 was detected using anti-myc, NSE4a was visualized using protein-S-HRP conjugate and the MAGEA1 protein was detected using anti-MAGEA1 antibody. Note that the endogenous levels of MAGEA1 in HEK293T cells are very low (lanes 4–6) compared to the levels of ectopically expressed MAGEA1 (lanes 1–3). These data suggest that TRIM31 binds directly to NSE4a and forms TRIM31-MAGEA1-NSE4a complex. ( B ) Cartoon comparing common features of the NSE1-NSE3-NSE4 and the TRIM31-MAGEA1-NSE4 complexes. The NSE3/MAGE proteins share conserved hydrophobic pockets (within the WH/B subdomain) interacting with NSE4/EID proteins. NSE3/MAGEG1 (WH/A) binds to NSE1 and stimulates (arrow) its RING-finger (RING) E3 ubiquitin-ligase activity. Similarly, MAGEA1 binds to TRIM31 and stimulates (arrow) its RING-finger (RING) E3 ubiquitin-ligase activity. Both, NSE3/MAGEG1 and MAGEA1, require their WH/A binding motifs for their stimulatory effects (arrowheads). As the TRIM31-MAGEA1-NSE4 complex shares common features with the yeast and human NSE1-NSE3-NSE4 complexes we propose that they may have evolved from a common ancestral NSE1-NSE3-NSE4 complex.

    Article Snippet: The amount of S-tagged proteins was analyzed on western blots using protein-S-HRP conjugate (Merck Millipore, http://www.emdmillipore.com/life-science-research/s-protein-hrp-conjugate/EMD_BIO-69047 ).

    Techniques: Construct, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Binding Assay