streptoavidin-conjugated peroxidase Millipore Search Results


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  • 95
    Millipore avidin peroxidase
    Avidin Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore streptavidin conjugated peroxidase
    Semaphorin 3A specifically binds sAPPα. (A) Wells in a 96-well plate were coated with either 50 ng sAPPα 695 (APP), 1 µg BSA or 50 ng Semaphorin 3A (Sema3A), blocked with 1% BSA and overlayed with different concentrations of Sema3A or sAPPα 695 (as indicated below the lanes). After washing, binding was determined by probing with anti-Sema3A antibody. The graph shows concentration-dependent binding of Sema3A to sAPPα 695 by densitometric quantification of the immunodots using NIH Image J. Bars represent averages of three independent experiments performed in triplicate each. The blot illustrates a representative experiment. (B) Wells in a 96-well plate were coated with 100 ng sAPPα 695 (APP), blocked with 1% BSA and incubated with increasing volumes of conditioned medium containing alkaline phosphatase-conjugated Semaphorin 3A (Sema3A-AP). After washing, binding was determined by measuring alkaline phosphatase activity. Bars represent averages of three independent experiments performed in triplicate. (C) <t>Streptavidin-coated</t> beads were incubated with biotinylated sAPPα 695 (bAPP) and Sema3A-AP-conditioned medium or control (non-transfected) medium. After washing, binding was evaluated by measuring alkaline phosphatase activity associated with the beads. Bars represent averages of three independent experiments.
    Streptavidin Conjugated Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore streptavidin horseradish peroxidase polymer conjugate
    Inhibition of biotin- G-S 59-1553 or biotin- G-S 470-1553 binding to fibronectin by unlabeled ShdA passenger domain or truncated peptides. (A) Thirty-three nanomolar biotinylated G-S 59-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 59-1553 (solid triangles), GST (solid squares), G-S 59-480 (solid circles), or G-S 470-1553 (open circles). (B) Thirty-three nanomolar biotinylated G-S 470-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 480-581 (solid triangles); G-S 602-1048 (open squares); G-S 1086-1553 (solid circles); G-S 470-1553 (open circles); a combination of G-S 480-581 , G-S 602-1048 , and G-S 1086-1553 (diamonds); or GST (solid squares). In each case, biotin associated with each well following washing was determined by ELISA using <t>streptavidin</t> conjugated with horseradish peroxidase. The mean A 410 values from six identical wells ± standard errors, expressed as percentages of values for control wells lacking added test peptides, are plotted.
    Streptavidin Horseradish Peroxidase Polymer Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore avidin conjugated to extravidin
    Inhibition of biotin- G-S 59-1553 or biotin- G-S 470-1553 binding to fibronectin by unlabeled ShdA passenger domain or truncated peptides. (A) Thirty-three nanomolar biotinylated G-S 59-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 59-1553 (solid triangles), GST (solid squares), G-S 59-480 (solid circles), or G-S 470-1553 (open circles). (B) Thirty-three nanomolar biotinylated G-S 470-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 480-581 (solid triangles); G-S 602-1048 (open squares); G-S 1086-1553 (solid circles); G-S 470-1553 (open circles); a combination of G-S 480-581 , G-S 602-1048 , and G-S 1086-1553 (diamonds); or GST (solid squares). In each case, biotin associated with each well following washing was determined by ELISA using <t>streptavidin</t> conjugated with horseradish peroxidase. The mean A 410 values from six identical wells ± standard errors, expressed as percentages of values for control wells lacking added test peptides, are plotted.
    Avidin Conjugated To Extravidin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore peroxidase
    Inhibition of biotin- G-S 59-1553 or biotin- G-S 470-1553 binding to fibronectin by unlabeled ShdA passenger domain or truncated peptides. (A) Thirty-three nanomolar biotinylated G-S 59-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 59-1553 (solid triangles), GST (solid squares), G-S 59-480 (solid circles), or G-S 470-1553 (open circles). (B) Thirty-three nanomolar biotinylated G-S 470-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 480-581 (solid triangles); G-S 602-1048 (open squares); G-S 1086-1553 (solid circles); G-S 470-1553 (open circles); a combination of G-S 480-581 , G-S 602-1048 , and G-S 1086-1553 (diamonds); or GST (solid squares). In each case, biotin associated with each well following washing was determined by ELISA using <t>streptavidin</t> conjugated with horseradish peroxidase. The mean A 410 values from six identical wells ± standard errors, expressed as percentages of values for control wells lacking added test peptides, are plotted.
    Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    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: 95/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore color conjugated streptavidin peroxidase
    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.
    Color Conjugated Streptavidin Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore streptavidin peroxidase conjugate
    Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with <t>streptavidin-peroxidase</t> conjugate and enhanced chemiluminescence.
    Streptavidin Peroxidase Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 316 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore horseradish rabbit peroxidase hrp conjugated streptavidin
    Cell surface glycan profiles of Ct and HeLa cells. ( A ) Lectin blot analysis of whole-cell lysates of Ct infectious forms (EBs) detected with biotinylated lectins followed by <t>streptavidin-HRP.</t> ( B ) Western blot analysis of MOMP and OmcB expression in whole bacterial cell lysates. ( C ) Glycophenotype of HeLa cells detected with biotinylated lectins and PE-conjugated streptavidin (filled curve) or incubated with PE-conjugated streptavidin alone (control; empty curve) analyzed by flow cytometry. In A and B blots are representative of three independent experiments. In C histograms are representative of three independent experiments.
    Horseradish Rabbit Peroxidase Hrp Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore avidin biotin peroxidase conjugated secondary antibody
    The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA <t>antibody.</t> Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as <t>secondary</t> antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. <t>HRP-conjugated</t> Streptavidin was used as a secondary antibody to detect <t>biotin-labeled</t> mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.
    Avidin Biotin Peroxidase Conjugated Secondary Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore hrp conjugated streptavidin
    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 <t>streptavidin-Cy3</t> (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 <t>anti-His-HRP</t> (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.
    Hrp Conjugated Streptavidin, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore peroxidase labeled streptavidin conjugate
    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 <t>streptavidin-Cy3</t> (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 <t>anti-His-HRP</t> (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.
    Peroxidase Labeled Streptavidin Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore avidin horseradish peroxidase conjugate
    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 <t>streptavidin-Cy3</t> (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 <t>anti-His-HRP</t> (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.
    Avidin Horseradish Peroxidase Conjugate, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore polyclonal goat anti mouse igg
    Kinase-active Lck is associated with the presence of a ZAP-70–pp21ζ complex and permissive anti–TCR-α/β signaling . ( A ) CD4 + primary lymph node T cells were purified as described in Materials and Methods. Lysate derived from 4 × 10 6 cell equivalents was subjected to four sequential precipitations with mAb specific for CD4, followed by three sequential precipitations with <t>polyclonal</t> anti-Lck. Precipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, blocked, probed with polyclonal anti-Lck, followed with HRP-conjugated Protein A, and developed using DuPont EC L reagents. ( B ) CD4 + lymph node T cells were purified as described in Materials and Methods. Immune complex kinase assays were performed as described in Materials and Methods, on precipitates derived from lysate containing 4 × 10 6 cell equivalents with mAbs specific for CD4, TCRCβ, and polyclonal anti-Lck. ( C ) The top row shows the pp21 signal associated with anti–ZAP-70 precipitates from lysates (5 × 10 6 cell equivalents) derived from primary CD4 + lymph node T cells, and clone 2.5.2 cultured in the presence ( IL-2 ) or absence ( REST ) of rIL-2. Anti–ZAP-70 precipitates were resolved on 12.5% SDS-PAGE, transferred to nitrocellulose, probed with 4G10 mAb specific for phosphotyrosine, developed with goat anti–mouse HRP-conjugated <t>IgG,</t> and revealed with DuPont EC L reagents. The middle row shows the pp21ζ signal associated with the anti–ZAP-70 precipitates. The blot shown in the top row was stripped and reprobed with ζ chain–specific mAb. The bottom row shows the same series of precipitations as in the top row, but immunoblotted with polyclonal anti–ZAP-70, developed with HRP-conjugated Protein A, and revealed with DuPont EC L reagents.
    Polyclonal Goat Anti Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore avidin peroxidase staining kit
    Kinase-active Lck is associated with the presence of a ZAP-70–pp21ζ complex and permissive anti–TCR-α/β signaling . ( A ) CD4 + primary lymph node T cells were purified as described in Materials and Methods. Lysate derived from 4 × 10 6 cell equivalents was subjected to four sequential precipitations with mAb specific for CD4, followed by three sequential precipitations with <t>polyclonal</t> anti-Lck. Precipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, blocked, probed with polyclonal anti-Lck, followed with HRP-conjugated Protein A, and developed using DuPont EC L reagents. ( B ) CD4 + lymph node T cells were purified as described in Materials and Methods. Immune complex kinase assays were performed as described in Materials and Methods, on precipitates derived from lysate containing 4 × 10 6 cell equivalents with mAbs specific for CD4, TCRCβ, and polyclonal anti-Lck. ( C ) The top row shows the pp21 signal associated with anti–ZAP-70 precipitates from lysates (5 × 10 6 cell equivalents) derived from primary CD4 + lymph node T cells, and clone 2.5.2 cultured in the presence ( IL-2 ) or absence ( REST ) of rIL-2. Anti–ZAP-70 precipitates were resolved on 12.5% SDS-PAGE, transferred to nitrocellulose, probed with 4G10 mAb specific for phosphotyrosine, developed with goat anti–mouse HRP-conjugated <t>IgG,</t> and revealed with DuPont EC L reagents. The middle row shows the pp21ζ signal associated with the anti–ZAP-70 precipitates. The blot shown in the top row was stripped and reprobed with ζ chain–specific mAb. The bottom row shows the same series of precipitations as in the top row, but immunoblotted with polyclonal anti–ZAP-70, developed with HRP-conjugated Protein A, and revealed with DuPont EC L reagents.
    Avidin Peroxidase Staining Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore horseradish peroxidase conjugated streptavidin
    HopF2 ADP-Ribosylates MKK5 in Vitro. (A) HopF2 inactivates MKK5 kinase activity in vitro. FLAG-MKK5 DD was coexpressed with GST or GST-tagged wild-type HopF2, HopF2 R71A , or HopF2 D175A in E. coli and purified with anti-FLAG M2-agarose. The activity of FLAG-MKK5 DD was measured by its ability to phosphorylate MPK6-His in vitro, as indicated by autoradiography. Amounts of protein were detected by immunoblot analysis with anti-FLAG and anti-MPK6 antibodies. (B) HopF2 ADP-ribosylates MKK5 in vitro. Purified HopF2, HopF2 R71A , or HopF2 D175A was incubated with purified FLAG-MKK5 DD or GST-MKK5 DD at a substrate-to-enzyme ratio of 10:1 in the presence of biotinylated NAD. ADP-ribosylation was detected by immunoblot using horseradish peroxidase–conjugated <t>streptavidin.</t> Amounts of MKK5 DD protein are indicated by CBB staining. (C) MKK5 DD is a better substrate for HopF2 than is WT MKK5. FLAG-tagged MKK5 DD or WT MKK5 was incubated with HopF2 or HopF2 D175A in the presence of biotinylated NAD, and ADP-ribosylation was detected as in (B) . Amounts of MKK5 protein are indicated by Coomassie blue staining. (D) HopF2 ADP-ribosylates MKK5 in a NAD concentration–dependent manner. Purified HopF2 or HopF2 D175A was incubated with FLAG-MKK5 DD in the ADP-RT reaction system containing the indicated concentrations of biotinylated NAD.
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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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    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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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with <t>streptavidin-HRP</t> and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input
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    Adhesion experiments. (a) <t>Streptavidin</t> binding to coated peptide. Data for 1:10,000 streptavidin dilution is shown: similar results were obtained at 1:3000 and 1:1000 dilutions. (b) Wild type and FcR-gamma knockout mice platelet adhesion to immulon-2B 96-well plates treated with various biotinylated and non-biotinylated peptides.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    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 <t>to</t> 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 <t>biotin-conjugated</t> 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 <t>anti-human</t> 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.
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    Image Search Results


    Semaphorin 3A specifically binds sAPPα. (A) Wells in a 96-well plate were coated with either 50 ng sAPPα 695 (APP), 1 µg BSA or 50 ng Semaphorin 3A (Sema3A), blocked with 1% BSA and overlayed with different concentrations of Sema3A or sAPPα 695 (as indicated below the lanes). After washing, binding was determined by probing with anti-Sema3A antibody. The graph shows concentration-dependent binding of Sema3A to sAPPα 695 by densitometric quantification of the immunodots using NIH Image J. Bars represent averages of three independent experiments performed in triplicate each. The blot illustrates a representative experiment. (B) Wells in a 96-well plate were coated with 100 ng sAPPα 695 (APP), blocked with 1% BSA and incubated with increasing volumes of conditioned medium containing alkaline phosphatase-conjugated Semaphorin 3A (Sema3A-AP). After washing, binding was determined by measuring alkaline phosphatase activity. Bars represent averages of three independent experiments performed in triplicate. (C) Streptavidin-coated beads were incubated with biotinylated sAPPα 695 (bAPP) and Sema3A-AP-conditioned medium or control (non-transfected) medium. After washing, binding was evaluated by measuring alkaline phosphatase activity associated with the beads. Bars represent averages of three independent experiments.

    Journal: PLoS ONE

    Article Title: Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

    doi: 10.1371/journal.pone.0022857

    Figure Lengend Snippet: Semaphorin 3A specifically binds sAPPα. (A) Wells in a 96-well plate were coated with either 50 ng sAPPα 695 (APP), 1 µg BSA or 50 ng Semaphorin 3A (Sema3A), blocked with 1% BSA and overlayed with different concentrations of Sema3A or sAPPα 695 (as indicated below the lanes). After washing, binding was determined by probing with anti-Sema3A antibody. The graph shows concentration-dependent binding of Sema3A to sAPPα 695 by densitometric quantification of the immunodots using NIH Image J. Bars represent averages of three independent experiments performed in triplicate each. The blot illustrates a representative experiment. (B) Wells in a 96-well plate were coated with 100 ng sAPPα 695 (APP), blocked with 1% BSA and incubated with increasing volumes of conditioned medium containing alkaline phosphatase-conjugated Semaphorin 3A (Sema3A-AP). After washing, binding was determined by measuring alkaline phosphatase activity. Bars represent averages of three independent experiments performed in triplicate. (C) Streptavidin-coated beads were incubated with biotinylated sAPPα 695 (bAPP) and Sema3A-AP-conditioned medium or control (non-transfected) medium. After washing, binding was evaluated by measuring alkaline phosphatase activity associated with the beads. Bars represent averages of three independent experiments.

    Article Snippet: The wells were washed six times with PBS, incubated with horseradish peroxidase-conjugated streptavidin (Sigma) for 1 h and developed with SuperSignal ELISA Femto Substrate.

    Techniques: Binding Assay, Concentration Assay, Incubation, Activity Assay, Transfection

    Inhibition of biotin- G-S 59-1553 or biotin- G-S 470-1553 binding to fibronectin by unlabeled ShdA passenger domain or truncated peptides. (A) Thirty-three nanomolar biotinylated G-S 59-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 59-1553 (solid triangles), GST (solid squares), G-S 59-480 (solid circles), or G-S 470-1553 (open circles). (B) Thirty-three nanomolar biotinylated G-S 470-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 480-581 (solid triangles); G-S 602-1048 (open squares); G-S 1086-1553 (solid circles); G-S 470-1553 (open circles); a combination of G-S 480-581 , G-S 602-1048 , and G-S 1086-1553 (diamonds); or GST (solid squares). In each case, biotin associated with each well following washing was determined by ELISA using streptavidin conjugated with horseradish peroxidase. The mean A 410 values from six identical wells ± standard errors, expressed as percentages of values for control wells lacking added test peptides, are plotted.

    Journal: Journal of Bacteriology

    Article Title: Fibronectin Binding to the Salmonella enterica Serotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat

    doi: 10.1128/JB.186.15.4931-4939.2004

    Figure Lengend Snippet: Inhibition of biotin- G-S 59-1553 or biotin- G-S 470-1553 binding to fibronectin by unlabeled ShdA passenger domain or truncated peptides. (A) Thirty-three nanomolar biotinylated G-S 59-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 59-1553 (solid triangles), GST (solid squares), G-S 59-480 (solid circles), or G-S 470-1553 (open circles). (B) Thirty-three nanomolar biotinylated G-S 470-1553 was incubated in wells coated with fibronectin (0.25 μg) with increasing amounts of G-S 480-581 (solid triangles); G-S 602-1048 (open squares); G-S 1086-1553 (solid circles); G-S 470-1553 (open circles); a combination of G-S 480-581 , G-S 602-1048 , and G-S 1086-1553 (diamonds); or GST (solid squares). In each case, biotin associated with each well following washing was determined by ELISA using streptavidin conjugated with horseradish peroxidase. The mean A 410 values from six identical wells ± standard errors, expressed as percentages of values for control wells lacking added test peptides, are plotted.

    Article Snippet: Alternatively, biotinylated protein ligand was detected with 1/1,000 streptavidin-horseradish peroxidase polymer conjugate (Sigma) in PBS, pH 7.4, and 0.04% Tween 20.

    Techniques: Inhibition, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay

    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

    Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with streptavidin-peroxidase conjugate and enhanced chemiluminescence.

    Journal: Applied and Environmental Microbiology

    Article Title: Dual Resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa Toxins in Heliothis virescens Suggests Multiple Mechanisms of Resistance

    doi: 10.1128/AEM.69.10.5898-5906.2003

    Figure Lengend Snippet: Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with streptavidin-peroxidase conjugate and enhanced chemiluminescence.

    Article Snippet: After blocking for 1 h in PBS plus 0.1% Tween 20 containing 3% BSA, membranes were incubated with streptavidin-peroxidase conjugate (Sigma) in PBS plus 0.1% Tween 20 plus 0.1% BSA for 1 h. After washing, biotinylated toxins were visualized using ECL (Amersham-Pharmacia) reagents following the manufacturer's instructions.

    Techniques: Binding Assay, Incubation, Centrifugation, Polyacrylamide Gel Electrophoresis

    Cell surface glycan profiles of Ct and HeLa cells. ( A ) Lectin blot analysis of whole-cell lysates of Ct infectious forms (EBs) detected with biotinylated lectins followed by streptavidin-HRP. ( B ) Western blot analysis of MOMP and OmcB expression in whole bacterial cell lysates. ( C ) Glycophenotype of HeLa cells detected with biotinylated lectins and PE-conjugated streptavidin (filled curve) or incubated with PE-conjugated streptavidin alone (control; empty curve) analyzed by flow cytometry. In A and B blots are representative of three independent experiments. In C histograms are representative of three independent experiments.

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

    Article Title: Glycosylation-dependent galectin–receptor interactions promote Chlamydia trachomatis infection

    doi: 10.1073/pnas.1802188115

    Figure Lengend Snippet: Cell surface glycan profiles of Ct and HeLa cells. ( A ) Lectin blot analysis of whole-cell lysates of Ct infectious forms (EBs) detected with biotinylated lectins followed by streptavidin-HRP. ( B ) Western blot analysis of MOMP and OmcB expression in whole bacterial cell lysates. ( C ) Glycophenotype of HeLa cells detected with biotinylated lectins and PE-conjugated streptavidin (filled curve) or incubated with PE-conjugated streptavidin alone (control; empty curve) analyzed by flow cytometry. In A and B blots are representative of three independent experiments. In C histograms are representative of three independent experiments.

    Article Snippet: Immunoreactivity was visualized using horseradish rabbit peroxidase (HRP)-conjugated streptavidin (Sigma) and Pierce ECL Western blotting substrate (Thermo Fisher Scientific Inc) with an ImageQuant LAS 4000 (GE Healthcare Life Science).

    Techniques: Western Blot, Expressing, Incubation, Flow Cytometry, Cytometry

    The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

    Journal: PLoS ONE

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    doi: 10.1371/journal.pone.0116237

    Figure Lengend Snippet: The mAbs recognize an epitope that contains an O-linked glycan modification of the octapeptide K-E/A-P-A-P-T-T-T/A/P. Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

    Article Snippet: Detection of the avidin/biotin/peroxidase-conjugated secondary antibody was performed using DAB FastTabs substrate (Sigma-Aldrich) for 5 minutes, followed by counterstaining with hematoxylin or hematoxylin and eosin.

    Techniques: Modification, Expressing, Recombinant, SDS Page, Mutagenesis, Variant Assay, Labeling

    mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA. (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.

    Journal: PLoS ONE

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    doi: 10.1371/journal.pone.0116237

    Figure Lengend Snippet: mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA. (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.

    Article Snippet: Detection of the avidin/biotin/peroxidase-conjugated secondary antibody was performed using DAB FastTabs substrate (Sigma-Aldrich) for 5 minutes, followed by counterstaining with hematoxylin or hematoxylin and eosin.

    Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, Concentration Assay, Recombinant

    The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).

    Journal: PLoS ONE

    Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints

    doi: 10.1371/journal.pone.0116237

    Figure Lengend Snippet: The mAbs detect an octapeptide motif present in the first mucin-like domain of human lubricin. Different domains of lubricin (depicted in the top panel beneath a schematic of the protein and its 12 coding exons) were cloned into a mammalian expression construct downstream of a signal peptide sequence and a Hemaglutinin epitope-tag (HA) sequence. Domain Mu1b was also fused to a human IgG-Fc fragment (Mu1b-Fc). An octapeptide motif (KEPAPTTT), which occurs multiple times within the first mucin-like domain (Mu1), was also fused to the IgG-Fc fragment (T-Fc). These constructs were transiently transfected into 293T cells. Serum-free conditioned media were collected and subjected to SDS-PAGE on 4–20% gradient gels. Anti-HA antibody detected all recombinant proteins in the media (HA), although weak immunodetectable bands occurred for Mu1a and Mu1c. Monoclonal antibodies 9g3, 7h12, 5c11 and 6a8 detected the secreted first mucin domain Mu1, Mu1a and Mu1c, but not Mu1b or the second mucin domain Mu2. Mu1b is the first 98 AA of the first mucin domain and does not contain KEPAPTTT; instead it has “KEPTPTT” and other “ETTT”-containing repeats. The only peptide motifs that Mu1a and Mu1c share is: “KEPAPTTP”. Antibodies 9g3, 7h12 and 6a8 also strongly recognized the KEPAPTTT containing recombinant protein (T-Fc), while 5c11 weakly interacted with T-Fc. Horseradish peroxidase (HRP) conjugated anti-mouse IgG, which was used as secondary antibody to detect 9g3, showed weak cross reactivity to human IgG-Fc. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled 7h12, 5c11 and 6a8, and showed no cross-reactivity by itself (data not shown).

    Article Snippet: Detection of the avidin/biotin/peroxidase-conjugated secondary antibody was performed using DAB FastTabs substrate (Sigma-Aldrich) for 5 minutes, followed by counterstaining with hematoxylin or hematoxylin and eosin.

    Techniques: Clone Assay, Expressing, Construct, Sequencing, Transfection, SDS Page, Recombinant, Labeling

    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

    Antigen capture by the MP65/bglu mAb. Panel A and B. Dose-response, β-glucan and MP65p capture curves. An Fc-specific, goat anti mouse IgG coating was used for the oriented binding of the bsmAb, at various concentrations, to the ELISA plates. Plate-bound bsmAb was then reacted with different doses of biotinylated laminarin (Panel A) or MP65p (Panel B). The amount of captured antigens was revealed by the addition of peroxidase-conjugated streptavidin followed by the enzyme substrate. O.D. 450 nm readings are the mean from duplicate wells after subtraction of O.D. values from the negative controls (wells with no antigen or no bsmAbs). R 2 values of the resulting capture curves were calculated by linear regression analysis. Panel C. Dual antigen capture by the bsmAb. The ELISA plate-bound bsmAb (0.2 μgml) was reacted in comparison with different doses of biotinylated laminarin or biotinylated MP65p or with a mixture containing both biotynilated laminarin and MP65p. The ELISA assay was performed as described above for data in Panels A and B.

    Journal: PLoS ONE

    Article Title: A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing β-Glucan and MP65 Determinants in Candida Species

    doi: 10.1371/journal.pone.0148714

    Figure Lengend Snippet: Antigen capture by the MP65/bglu mAb. Panel A and B. Dose-response, β-glucan and MP65p capture curves. An Fc-specific, goat anti mouse IgG coating was used for the oriented binding of the bsmAb, at various concentrations, to the ELISA plates. Plate-bound bsmAb was then reacted with different doses of biotinylated laminarin (Panel A) or MP65p (Panel B). The amount of captured antigens was revealed by the addition of peroxidase-conjugated streptavidin followed by the enzyme substrate. O.D. 450 nm readings are the mean from duplicate wells after subtraction of O.D. values from the negative controls (wells with no antigen or no bsmAbs). R 2 values of the resulting capture curves were calculated by linear regression analysis. Panel C. Dual antigen capture by the bsmAb. The ELISA plate-bound bsmAb (0.2 μgml) was reacted in comparison with different doses of biotinylated laminarin or biotinylated MP65p or with a mixture containing both biotynilated laminarin and MP65p. The ELISA assay was performed as described above for data in Panels A and B.

    Article Snippet: Wells were washed again and the amount of bound antigen was evaluated by the addition of horse-radish peroxidase-conjugated streptavidin (Sigma-Aldrich) followed by 3,3′,5,5′-tetramethylbenzidine-H2 O2 substrate solution (1-step Ultra-TMB ELISA, Thermo Scientific, USA).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

    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

    Kinase-active Lck is associated with the presence of a ZAP-70–pp21ζ complex and permissive anti–TCR-α/β signaling . ( A ) CD4 + primary lymph node T cells were purified as described in Materials and Methods. Lysate derived from 4 × 10 6 cell equivalents was subjected to four sequential precipitations with mAb specific for CD4, followed by three sequential precipitations with polyclonal anti-Lck. Precipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, blocked, probed with polyclonal anti-Lck, followed with HRP-conjugated Protein A, and developed using DuPont EC L reagents. ( B ) CD4 + lymph node T cells were purified as described in Materials and Methods. Immune complex kinase assays were performed as described in Materials and Methods, on precipitates derived from lysate containing 4 × 10 6 cell equivalents with mAbs specific for CD4, TCRCβ, and polyclonal anti-Lck. ( C ) The top row shows the pp21 signal associated with anti–ZAP-70 precipitates from lysates (5 × 10 6 cell equivalents) derived from primary CD4 + lymph node T cells, and clone 2.5.2 cultured in the presence ( IL-2 ) or absence ( REST ) of rIL-2. Anti–ZAP-70 precipitates were resolved on 12.5% SDS-PAGE, transferred to nitrocellulose, probed with 4G10 mAb specific for phosphotyrosine, developed with goat anti–mouse HRP-conjugated IgG, and revealed with DuPont EC L reagents. The middle row shows the pp21ζ signal associated with the anti–ZAP-70 precipitates. The blot shown in the top row was stripped and reprobed with ζ chain–specific mAb. The bottom row shows the same series of precipitations as in the top row, but immunoblotted with polyclonal anti–ZAP-70, developed with HRP-conjugated Protein A, and revealed with DuPont EC L reagents.

    Journal: The Journal of Experimental Medicine

    Article Title: Interleukin 2-mediated Uncoupling of T Cell Receptor ?/? from CD3 Signaling

    doi:

    Figure Lengend Snippet: Kinase-active Lck is associated with the presence of a ZAP-70–pp21ζ complex and permissive anti–TCR-α/β signaling . ( A ) CD4 + primary lymph node T cells were purified as described in Materials and Methods. Lysate derived from 4 × 10 6 cell equivalents was subjected to four sequential precipitations with mAb specific for CD4, followed by three sequential precipitations with polyclonal anti-Lck. Precipitates were resolved by 8% SDS-PAGE, transferred to nitrocellulose, blocked, probed with polyclonal anti-Lck, followed with HRP-conjugated Protein A, and developed using DuPont EC L reagents. ( B ) CD4 + lymph node T cells were purified as described in Materials and Methods. Immune complex kinase assays were performed as described in Materials and Methods, on precipitates derived from lysate containing 4 × 10 6 cell equivalents with mAbs specific for CD4, TCRCβ, and polyclonal anti-Lck. ( C ) The top row shows the pp21 signal associated with anti–ZAP-70 precipitates from lysates (5 × 10 6 cell equivalents) derived from primary CD4 + lymph node T cells, and clone 2.5.2 cultured in the presence ( IL-2 ) or absence ( REST ) of rIL-2. Anti–ZAP-70 precipitates were resolved on 12.5% SDS-PAGE, transferred to nitrocellulose, probed with 4G10 mAb specific for phosphotyrosine, developed with goat anti–mouse HRP-conjugated IgG, and revealed with DuPont EC L reagents. The middle row shows the pp21ζ signal associated with the anti–ZAP-70 precipitates. The blot shown in the top row was stripped and reprobed with ζ chain–specific mAb. The bottom row shows the same series of precipitations as in the top row, but immunoblotted with polyclonal anti–ZAP-70, developed with HRP-conjugated Protein A, and revealed with DuPont EC L reagents.

    Article Snippet: 4G10 immunoblots were developed with horseradish peroxidase (HRP)1 -conjugated polyclonal goat anti–mouse IgG ( Sigma Chemical Co. , St. Louis, MO), in 1% gelatin (Bio- Rad, Hercules, CA).

    Techniques: Purification, Derivative Assay, SDS Page, Immune Complex Kinase Assay, Cell Culture

    HopF2 ADP-Ribosylates MKK5 in Vitro. (A) HopF2 inactivates MKK5 kinase activity in vitro. FLAG-MKK5 DD was coexpressed with GST or GST-tagged wild-type HopF2, HopF2 R71A , or HopF2 D175A in E. coli and purified with anti-FLAG M2-agarose. The activity of FLAG-MKK5 DD was measured by its ability to phosphorylate MPK6-His in vitro, as indicated by autoradiography. Amounts of protein were detected by immunoblot analysis with anti-FLAG and anti-MPK6 antibodies. (B) HopF2 ADP-ribosylates MKK5 in vitro. Purified HopF2, HopF2 R71A , or HopF2 D175A was incubated with purified FLAG-MKK5 DD or GST-MKK5 DD at a substrate-to-enzyme ratio of 10:1 in the presence of biotinylated NAD. ADP-ribosylation was detected by immunoblot using horseradish peroxidase–conjugated streptavidin. Amounts of MKK5 DD protein are indicated by CBB staining. (C) MKK5 DD is a better substrate for HopF2 than is WT MKK5. FLAG-tagged MKK5 DD or WT MKK5 was incubated with HopF2 or HopF2 D175A in the presence of biotinylated NAD, and ADP-ribosylation was detected as in (B) . Amounts of MKK5 protein are indicated by Coomassie blue staining. (D) HopF2 ADP-ribosylates MKK5 in a NAD concentration–dependent manner. Purified HopF2 or HopF2 D175A was incubated with FLAG-MKK5 DD in the ADP-RT reaction system containing the indicated concentrations of biotinylated NAD.

    Journal: The Plant Cell

    Article Title: A Pseudomonas syringae ADP-Ribosyltransferase Inhibits Arabidopsis Mitogen-Activated Protein Kinase Kinases [W]

    doi: 10.1105/tpc.110.075697

    Figure Lengend Snippet: HopF2 ADP-Ribosylates MKK5 in Vitro. (A) HopF2 inactivates MKK5 kinase activity in vitro. FLAG-MKK5 DD was coexpressed with GST or GST-tagged wild-type HopF2, HopF2 R71A , or HopF2 D175A in E. coli and purified with anti-FLAG M2-agarose. The activity of FLAG-MKK5 DD was measured by its ability to phosphorylate MPK6-His in vitro, as indicated by autoradiography. Amounts of protein were detected by immunoblot analysis with anti-FLAG and anti-MPK6 antibodies. (B) HopF2 ADP-ribosylates MKK5 in vitro. Purified HopF2, HopF2 R71A , or HopF2 D175A was incubated with purified FLAG-MKK5 DD or GST-MKK5 DD at a substrate-to-enzyme ratio of 10:1 in the presence of biotinylated NAD. ADP-ribosylation was detected by immunoblot using horseradish peroxidase–conjugated streptavidin. Amounts of MKK5 DD protein are indicated by CBB staining. (C) MKK5 DD is a better substrate for HopF2 than is WT MKK5. FLAG-tagged MKK5 DD or WT MKK5 was incubated with HopF2 or HopF2 D175A in the presence of biotinylated NAD, and ADP-ribosylation was detected as in (B) . Amounts of MKK5 protein are indicated by Coomassie blue staining. (D) HopF2 ADP-ribosylates MKK5 in a NAD concentration–dependent manner. Purified HopF2 or HopF2 D175A was incubated with FLAG-MKK5 DD in the ADP-RT reaction system containing the indicated concentrations of biotinylated NAD.

    Article Snippet: The ADP-ribosylated MKK5DD proteins were analyzed by immunoblot with horseradish peroxidase–conjugated streptavidin (Sigma-Aldrich).

    Techniques: In Vitro, Activity Assay, Purification, Autoradiography, Incubation, Staining, Concentration Assay

    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

    ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with streptavidin-HRP and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input

    Journal: Journal of Inflammation (London, England)

    Article Title: Differential splicing of the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) regulates inflammasomes

    doi: 10.1186/1476-9255-7-23

    Figure Lengend Snippet: ASC and ASC-b interact with pro-caspase 1 with similar affinity . Immobilized GST-caspase 1-CARD was incubated with in vitro translated and biotinylated ASC or ASC-b and subjected to in vitro GST-pull down assays using GST-caspase 1-CARD and GST control immobilized to GSH Sepharose, as indicated. Bound proteins were visualized with streptavidin-HRP and ECL-Plus detection (Amersham Pharmacia Biotech). 10% of the in vitro translated proteins were loaded as 'input".

    Article Snippet: Bound proteins were washed 4 times in HKMEN buffer supplemented with protease inhibitors, boiled in Laemmli buffer, separated by SDS/PAGE, transferred onto a PVDF membrane, and detected with Streptavidin-HRP in conjugation with an enhanced chemiluminescent reagent (Millipore).

    Techniques: Incubation, In Vitro

    Adhesion experiments. (a) Streptavidin binding to coated peptide. Data for 1:10,000 streptavidin dilution is shown: similar results were obtained at 1:3000 and 1:1000 dilutions. (b) Wild type and FcR-gamma knockout mice platelet adhesion to immulon-2B 96-well plates treated with various biotinylated and non-biotinylated peptides.

    Journal: Peptides

    Article Title: The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

    doi: 10.1016/j.peptides.2012.04.013

    Figure Lengend Snippet: Adhesion experiments. (a) Streptavidin binding to coated peptide. Data for 1:10,000 streptavidin dilution is shown: similar results were obtained at 1:3000 and 1:1000 dilutions. (b) Wild type and FcR-gamma knockout mice platelet adhesion to immulon-2B 96-well plates treated with various biotinylated and non-biotinylated peptides.

    Article Snippet: Coated peptide was quantitated by adding 200 μL of a 1:10,000 dilution of a streptavidin–horseradish peroxidase solution (Chemicon), washing, and developing with 200 μL 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Scientific).

    Techniques: Binding Assay, Knock-Out, Mouse Assay

    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