anti-human igg-conjugated dynabead solution Search Results


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  • 99
    Vector Laboratories anti rabbit
    Anti Rabbit, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 2197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher magnetic sheep anti rat igg dynabeads
    Magnetic Sheep Anti Rat Igg Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg conjugated dynabeads
    Goat Anti Mouse Igg Conjugated Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti cd14 mouse igg
    Anti Cd14 Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads m 280 sheep anti rabbit igg
    Dynabeads M 280 Sheep Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 686 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit igg abs
    Isolation of α 4 nAChRs and their signaling partners in <t>CD4</t> + cells. (A) Western blot detection of α 4 and β 2 nAChRs, CD4, and CD16 within the α 4 + fraction isolated from ICF C using the bead assay ( n = 3 mice for WT and α 4 −/− ). <t>IgG</t> beads (+ lane) and ICF C from α 4 −/− mice (right lane) were used as controls. (B) FACS immunosorting of α 4 + cells from ICF C ( n = 3 mice). (C) The α 4 + population from B was separated using an anti-CD4 + Ab ( n = 3 mice). (D) Immunocytochemical detection of α 4 nAChR (green), CD4 (red), and CD3 (blue) within the ICF C . Fluorescent signals are overlaid on differential interference contrast images. The diagram shows the proportion of stained cells in the CD3 + ICF C ( n = 3 mice). Scale bar: 50 μ m. (E) A Coomassie-stained gel showing the position of bands (blue boxes) analyzed by LC-ESI MS within α 4 nAChR and Gprin1 IP experiments from ICF C . The α 4 −/− . Western blot confirmation of the interaction is shown in red boxes ( n = 3 mice). (F) Colocalization of α 4 nAChRs, Gprin1, and phalloidin (red) in a CD4 + cell isolated using the bead assay. The bottom image shows the expression of the proteins in actin-rich domains (arrow). Scale bar: 1 μ m.
    Rabbit Igg Abs, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protein g conjugated dynabeads
    Schematic illustration of two-step purification of affinity binder-oligonucleotide conjugates. (a) Antibody-DNA conjugates illustrated here as an example. Conjugation of antibodies and oligonucleotides yields a mixture of desired conjugates, along with unconjugated antibodies and unconjugated oligonucleotides. (A) Biotinylated capture DNA oligonucleotides are hybridized to the oligonucleotides in the mixture. (B) Streptavidin-coated Sepharose beads are used to capture the biotinylated capture oligonucleotides, both in the form of conjugates and free oligonucleotides. (C) The unconjugated antibodies are removed by washes. (D) The MlyI enzyme is used to cleave the captured oligonucleotide hybrids, allowing both conjugates and oligonucleotides to be eluted from the solid support. (E) The eluate is then incubated either with <t>protein</t> G beads (for antibody-DNA conjugates) or with <t>Dynabeads</t> His tag (for DARPin-DNA conjugates), while free oligonucleotides are removed by washes. (F) Finally, purified conjugates are eluted from the solid support. (b) Illustration of hybridization of Arm1_long and Arm1 Capture, and the subsequent MlyI cleavage.
    Protein G Conjugated Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher protein a conjugated dynabeads
    Schematic illustration of two-step purification of affinity binder-oligonucleotide conjugates. (a) Antibody-DNA conjugates illustrated here as an example. Conjugation of antibodies and oligonucleotides yields a mixture of desired conjugates, along with unconjugated antibodies and unconjugated oligonucleotides. (A) Biotinylated capture DNA oligonucleotides are hybridized to the oligonucleotides in the mixture. (B) Streptavidin-coated Sepharose beads are used to capture the biotinylated capture oligonucleotides, both in the form of conjugates and free oligonucleotides. (C) The unconjugated antibodies are removed by washes. (D) The MlyI enzyme is used to cleave the captured oligonucleotide hybrids, allowing both conjugates and oligonucleotides to be eluted from the solid support. (E) The eluate is then incubated either with <t>protein</t> G beads (for antibody-DNA conjugates) or with <t>Dynabeads</t> His tag (for DARPin-DNA conjugates), while free oligonucleotides are removed by washes. (F) Finally, purified conjugates are eluted from the solid support. (b) Illustration of hybridization of Arm1_long and Arm1 Capture, and the subsequent MlyI cleavage.
    Protein A Conjugated Dynabeads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bethyl anti human igd
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Anti Human Igd, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems mouse anti human cd9
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Mouse Anti Human Cd9, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rrid ab 10392384 rabbit polyclonal igg anti human atf 2 c 19 santa cruz biotechnology
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Rrid Ab 10392384 Rabbit Polyclonal Igg Anti Human Atf 2 C 19 Santa Cruz Biotechnology, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti rabbit igg
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Anti Rabbit Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 4314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti sheep igg whole molecule peroxidase
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Anti Sheep Igg Whole Molecule Peroxidase, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend rrid ab 314738 mouse monoclonal fitc anti human cd36
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Rrid Ab 314738 Mouse Monoclonal Fitc Anti Human Cd36, supplied by BioLegend, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore phosstop trypan blue solution
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Phosstop Trypan Blue Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher assays dynabeads untouched human monocytes negative isolation kit thermofisher scientific
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Assays Dynabeads Untouched Human Monocytes Negative Isolation Kit Thermofisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies igg
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Igg, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 2252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega human recombinant bfgf
    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ <t>(anti‐IgD;</t> dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK <t>Y204</t> (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.
    Human Recombinant Bfgf, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti mouse shp 1 ab
    Siglec-E expression on tissue macrophages and bone marrow-derived macrophages (BMDM) is upregulated following lipopolysaccharide (LPS) treatment. (A) Wild-type (WT), KO1, and R126D mice were injected with 15 µg LPS or PBS intraperitoneally. After 3 h, animals were euthanized and tissue samples collected and frozen. Liver cryostat sections were labeled with sheep anti-siglec-E Ab directly labeled with Alexa 488 and anti-siglec-1 Abs SER-4 and 3D6 followed by anti-rat Alexa 647. Sections were also stained with DAPI to reveal nuclei. Siglec-E is expressed on Kupffer cells, which co-express siglec-1, and is upregulated following LPS stimulation in WT but not KO1 or R126D mice. Green dots in the anti-siglec-E stained KO1 and R126D sections are due to non-specific binding of the antibody (Ab). The scale bar represents 10 µm. (B) Siglec-E is expressed at low levels on BMDM and strongly upregulated following 3 days culture in 1 ng/ml LPS. (C) Siglec-E is constitutively phosphorylated in LPS-stimulated BMDM. WT and KO2 BMDM were treated for 3 days with 1 ng/ml LPS and the upregulated siglec-E was immunoprecipitated (IP) using sheep ant-siglec-E Ab and immunoblotted (IB) for phosphotyrosine, <t>SHP-1</t> and siglec-E.
    Anti Mouse Shp 1 Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dynabeads protein immunoprecipitation kit
    Siglec-E expression on tissue macrophages and bone marrow-derived macrophages (BMDM) is upregulated following lipopolysaccharide (LPS) treatment. (A) Wild-type (WT), KO1, and R126D mice were injected with 15 µg LPS or PBS intraperitoneally. After 3 h, animals were euthanized and tissue samples collected and frozen. Liver cryostat sections were labeled with sheep anti-siglec-E Ab directly labeled with Alexa 488 and anti-siglec-1 Abs SER-4 and 3D6 followed by anti-rat Alexa 647. Sections were also stained with DAPI to reveal nuclei. Siglec-E is expressed on Kupffer cells, which co-express siglec-1, and is upregulated following LPS stimulation in WT but not KO1 or R126D mice. Green dots in the anti-siglec-E stained KO1 and R126D sections are due to non-specific binding of the antibody (Ab). The scale bar represents 10 µm. (B) Siglec-E is expressed at low levels on BMDM and strongly upregulated following 3 days culture in 1 ng/ml LPS. (C) Siglec-E is constitutively phosphorylated in LPS-stimulated BMDM. WT and KO2 BMDM were treated for 3 days with 1 ng/ml LPS and the upregulated siglec-E was immunoprecipitated (IP) using sheep ant-siglec-E Ab and immunoblotted (IB) for phosphotyrosine, <t>SHP-1</t> and siglec-E.
    Dynabeads Protein Immunoprecipitation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Isolation of α 4 nAChRs and their signaling partners in CD4 + cells. (A) Western blot detection of α 4 and β 2 nAChRs, CD4, and CD16 within the α 4 + fraction isolated from ICF C using the bead assay ( n = 3 mice for WT and α 4 −/− ). IgG beads (+ lane) and ICF C from α 4 −/− mice (right lane) were used as controls. (B) FACS immunosorting of α 4 + cells from ICF C ( n = 3 mice). (C) The α 4 + population from B was separated using an anti-CD4 + Ab ( n = 3 mice). (D) Immunocytochemical detection of α 4 nAChR (green), CD4 (red), and CD3 (blue) within the ICF C . Fluorescent signals are overlaid on differential interference contrast images. The diagram shows the proportion of stained cells in the CD3 + ICF C ( n = 3 mice). Scale bar: 50 μ m. (E) A Coomassie-stained gel showing the position of bands (blue boxes) analyzed by LC-ESI MS within α 4 nAChR and Gprin1 IP experiments from ICF C . The α 4 −/− . Western blot confirmation of the interaction is shown in red boxes ( n = 3 mice). (F) Colocalization of α 4 nAChRs, Gprin1, and phalloidin (red) in a CD4 + cell isolated using the bead assay. The bottom image shows the expression of the proteins in actin-rich domains (arrow). Scale bar: 1 μ m.

    Journal: Molecular Pharmacology

    Article Title: The α4 Nicotinic Receptor Promotes CD4+

    doi: 10.1124/mol.113.088484

    Figure Lengend Snippet: Isolation of α 4 nAChRs and their signaling partners in CD4 + cells. (A) Western blot detection of α 4 and β 2 nAChRs, CD4, and CD16 within the α 4 + fraction isolated from ICF C using the bead assay ( n = 3 mice for WT and α 4 −/− ). IgG beads (+ lane) and ICF C from α 4 −/− mice (right lane) were used as controls. (B) FACS immunosorting of α 4 + cells from ICF C ( n = 3 mice). (C) The α 4 + population from B was separated using an anti-CD4 + Ab ( n = 3 mice). (D) Immunocytochemical detection of α 4 nAChR (green), CD4 (red), and CD3 (blue) within the ICF C . Fluorescent signals are overlaid on differential interference contrast images. The diagram shows the proportion of stained cells in the CD3 + ICF C ( n = 3 mice). Scale bar: 50 μ m. (E) A Coomassie-stained gel showing the position of bands (blue boxes) analyzed by LC-ESI MS within α 4 nAChR and Gprin1 IP experiments from ICF C . The α 4 −/− . Western blot confirmation of the interaction is shown in red boxes ( n = 3 mice). (F) Colocalization of α 4 nAChRs, Gprin1, and phalloidin (red) in a CD4 + cell isolated using the bead assay. The bottom image shows the expression of the proteins in actin-rich domains (arrow). Scale bar: 1 μ m.

    Article Snippet: For cell isolation, the ICF was preincubated with 10 μ g of rat monoclonal anti- α 4 antibody (mAb299) , mouse polyclonal anti-CD4 anitbody (Ab) (Santa Cruz Biotechnology, Dallas, TX), or rabbit IgG Abs (Cell Signaling Technologies, Beverly, MA) in ice-cold PBS for 2 hours before the addition of a precleared Protein G Dynabeads resin (Invitrogen/Life Technologies, Carlsbad, CA).

    Techniques: Isolation, Western Blot, Mouse Assay, FACS, Staining, Mass Spectrometry, Expressing

    Schematic illustration of two-step purification of affinity binder-oligonucleotide conjugates. (a) Antibody-DNA conjugates illustrated here as an example. Conjugation of antibodies and oligonucleotides yields a mixture of desired conjugates, along with unconjugated antibodies and unconjugated oligonucleotides. (A) Biotinylated capture DNA oligonucleotides are hybridized to the oligonucleotides in the mixture. (B) Streptavidin-coated Sepharose beads are used to capture the biotinylated capture oligonucleotides, both in the form of conjugates and free oligonucleotides. (C) The unconjugated antibodies are removed by washes. (D) The MlyI enzyme is used to cleave the captured oligonucleotide hybrids, allowing both conjugates and oligonucleotides to be eluted from the solid support. (E) The eluate is then incubated either with protein G beads (for antibody-DNA conjugates) or with Dynabeads His tag (for DARPin-DNA conjugates), while free oligonucleotides are removed by washes. (F) Finally, purified conjugates are eluted from the solid support. (b) Illustration of hybridization of Arm1_long and Arm1 Capture, and the subsequent MlyI cleavage.

    Journal: PLoS ONE

    Article Title: A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis

    doi: 10.1371/journal.pone.0108061

    Figure Lengend Snippet: Schematic illustration of two-step purification of affinity binder-oligonucleotide conjugates. (a) Antibody-DNA conjugates illustrated here as an example. Conjugation of antibodies and oligonucleotides yields a mixture of desired conjugates, along with unconjugated antibodies and unconjugated oligonucleotides. (A) Biotinylated capture DNA oligonucleotides are hybridized to the oligonucleotides in the mixture. (B) Streptavidin-coated Sepharose beads are used to capture the biotinylated capture oligonucleotides, both in the form of conjugates and free oligonucleotides. (C) The unconjugated antibodies are removed by washes. (D) The MlyI enzyme is used to cleave the captured oligonucleotide hybrids, allowing both conjugates and oligonucleotides to be eluted from the solid support. (E) The eluate is then incubated either with protein G beads (for antibody-DNA conjugates) or with Dynabeads His tag (for DARPin-DNA conjugates), while free oligonucleotides are removed by washes. (F) Finally, purified conjugates are eluted from the solid support. (b) Illustration of hybridization of Arm1_long and Arm1 Capture, and the subsequent MlyI cleavage.

    Article Snippet: Next, for antibody-DNA conjugates, the supernatant from the MlyI cleavage was incubated with Dynabeads Protein G (10004D, Life Technologies) for 1 h at RT, followed by two washes with PBST to remove unconjugated oligonucleotides.

    Techniques: Purification, Conjugation Assay, Incubation, Hybridization

    Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ (anti‐IgD; dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK Y204 (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.

    Journal: Journal of Clinical Pharmacology

    Article Title: PRT062607 Achieves Complete Inhibition of the Spleen Tyrosine Kinase at Tolerated Exposures Following Oral Dosing in Healthy Volunteers

    doi: 10.1002/jcph.794

    Figure Lengend Snippet: Kinetics of SYK inhibition following a single oral dose of PRT062607. The percentage of predose levels of BCR/SYK‐ (anti‐IgD; dark gray bars) or PMA/PKC‐ (light gray bars) induced B‐cell activation (A), FcεR1/SYK‐ (anti‐IgE; dark gray bars) or fMLP‐ (light gray bars) induced basophil degranulation (B), and BCR/SYK‐mediated pERK Y204 (black bars), BCR/LYN‐mediated pSYK Y352 (light gray bars), and PMA/PKC‐mediated pERK Y204 (dark gray bars) (C) are shown on the first (left) y‐axis. The average PRT062607 plasma concentration (nM) is presented as a tracing and defined on the second (right) y‐axis. Hours postdose are depicted on the x‐axis. Error bars represent the standard error of the mean.

    Article Snippet: The following reagents were procured from other sources: ERK threonine (T) 202/Y204 alexafluor 488 (Cell Signaling Technologies, Danvers, Massachusetts), goat anti–human IgE and anti–human IgD (Bethyl Laboratories, Montgomery, Texas), CD3/CD28 Dynabeads (Life Technologies Corporation, Grand Island, New York), CD14 microbeads and MS columns for monocyte purification from whole blood (Miltenyi Biotech, Auburn, California), recombinant human IL4 and GM‐CSF (R & D Systems, Minneapolis, Minnesota), BasoTest and Phagoburst kits for measuring basophil degranulation and neutrophil oxidative burst, respectively (Orpegen Pharma, Heidelberg, Germany), lymphoprep solution for peripheral blood mononuclear cell isolation (Stem Cell Technologies, Vancouver, Canada), phosphate buffered saline (PBS), bovine serum albumin (BSA), phorbol 12‐myristate 13‐acetate (PMA), and lipopolysaccharide (LPS) were all obtained from Sigma‐Aldrich (St. Louis, Missouri).

    Techniques: Inhibition, Activation Assay, Concentration Assay

    Siglec-E expression on tissue macrophages and bone marrow-derived macrophages (BMDM) is upregulated following lipopolysaccharide (LPS) treatment. (A) Wild-type (WT), KO1, and R126D mice were injected with 15 µg LPS or PBS intraperitoneally. After 3 h, animals were euthanized and tissue samples collected and frozen. Liver cryostat sections were labeled with sheep anti-siglec-E Ab directly labeled with Alexa 488 and anti-siglec-1 Abs SER-4 and 3D6 followed by anti-rat Alexa 647. Sections were also stained with DAPI to reveal nuclei. Siglec-E is expressed on Kupffer cells, which co-express siglec-1, and is upregulated following LPS stimulation in WT but not KO1 or R126D mice. Green dots in the anti-siglec-E stained KO1 and R126D sections are due to non-specific binding of the antibody (Ab). The scale bar represents 10 µm. (B) Siglec-E is expressed at low levels on BMDM and strongly upregulated following 3 days culture in 1 ng/ml LPS. (C) Siglec-E is constitutively phosphorylated in LPS-stimulated BMDM. WT and KO2 BMDM were treated for 3 days with 1 ng/ml LPS and the upregulated siglec-E was immunoprecipitated (IP) using sheep ant-siglec-E Ab and immunoblotted (IB) for phosphotyrosine, SHP-1 and siglec-E.

    Journal: Frontiers in Immunology

    Article Title: Expression of Siglec-E Alters the Proteome of Lipopolysaccharide (LPS)-Activated Macrophages but Does Not Affect LPS-Driven Cytokine Production or Toll-Like Receptor 4 Endocytosis

    doi: 10.3389/fimmu.2017.01926

    Figure Lengend Snippet: Siglec-E expression on tissue macrophages and bone marrow-derived macrophages (BMDM) is upregulated following lipopolysaccharide (LPS) treatment. (A) Wild-type (WT), KO1, and R126D mice were injected with 15 µg LPS or PBS intraperitoneally. After 3 h, animals were euthanized and tissue samples collected and frozen. Liver cryostat sections were labeled with sheep anti-siglec-E Ab directly labeled with Alexa 488 and anti-siglec-1 Abs SER-4 and 3D6 followed by anti-rat Alexa 647. Sections were also stained with DAPI to reveal nuclei. Siglec-E is expressed on Kupffer cells, which co-express siglec-1, and is upregulated following LPS stimulation in WT but not KO1 or R126D mice. Green dots in the anti-siglec-E stained KO1 and R126D sections are due to non-specific binding of the antibody (Ab). The scale bar represents 10 µm. (B) Siglec-E is expressed at low levels on BMDM and strongly upregulated following 3 days culture in 1 ng/ml LPS. (C) Siglec-E is constitutively phosphorylated in LPS-stimulated BMDM. WT and KO2 BMDM were treated for 3 days with 1 ng/ml LPS and the upregulated siglec-E was immunoprecipitated (IP) using sheep ant-siglec-E Ab and immunoblotted (IB) for phosphotyrosine, SHP-1 and siglec-E.

    Article Snippet: Materials Dulbecco’s phosphate-buffered saline (PBS) without Ca and Mg, fetal bovine serum (FBS) (qualified, heat inactivated, E.U.-approved), penicillin and streptomycin solution, Trypsin–EDTA solution, protein G Dynabeads, Microplate BCA Protein Assay, NuPAGE LDS Sample Buffer, NuPAGE® Novex 4–12% Bis-Tris gel, MOPS running buffer, and sample reducing agent, trypsin protease, Pierce MS Grade, TMT 10-plex™ Isobaric Reagent Label Set were from Thermo Fisher Scientific, Paisley, UK; Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles were from GE LifeSciences; Roche-COMPLETE Mini EDTA-Free Protease Inhibitor tablets, Roche-PHOSS-RO, PhosSTOP™ Trypan blue solution, anti-sheep IgG (whole molecule)-peroxidase and anti-rabbit IgG (whole molecule)–peroxidase Ab produced in goat, lipopolysaccharide from E. coli 0111:B4 were from Sigma; GM-CSF and IL-4 were from Peprotech, GolgiStop, CD16/CD32 (Fc block), V500 rat anti-mouse I-A/I-E (clone: M5/114; 562366) were from BD Bioscience, UK; anti-mouse TNF alpha PE (clone: MP6-XT22), anti-mouse CD11c PE-cy7 (Clone: N418), anti-mouse Ly-6G (Gr-1) Alexa Fluor® 488 (clone: RB6-8C5) were from eBioscience, UK; anti-Salmonella Typhimurium (clone: 1E6), anti-phosphotyrosine Ab (HRP) (Abcam clone: PY20-ab16389) were from Abcam, UK; APC anti-mouse CD11c Ab (clone: N418), PE-conjugated anti-siglec-E used in flow cytometry (clone: M1304A01), biotin anti-mouse TLR4 (CD284)/MD2 complex Ab (clone: MTS510), PE/Cy7 anti-mouse TLR4 (CD284)/MD2 complex Ab (clone: MTS510), PE anti-mouse/human CD11b Ab (clone: M1/70), APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1) Ab (clone: RB6-8C5) were from Biolegend, UK; and anti-mouse SHP-1 Ab (clone: C-19) was from Santa Cruz.

    Techniques: Expressing, Derivative Assay, Mouse Assay, Injection, Labeling, Staining, Binding Assay, Immunoprecipitation