peroxidase-conjugated secondary antibody Search Results


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  • 99
    Millipore secondary peroxidase conjugated secondary antibodies
    Secondary Peroxidase Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc peroxidase conjugated secondary antibody
    Peroxidase Conjugated Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 2755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad horseradish peroxidaseconjugated secondary antibody
    Horseradish Peroxidaseconjugated Secondary Antibody, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc peroxidase conjugate secondary antibody
    Peroxidase Conjugate Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore peroxidase conjugated secondary antibodies
    Peroxidase Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare peroxidase conjugated secondary antibody
    Peroxidase Conjugated Secondary Antibody, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 7651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GeneTex peroxidase conjugated secondary antibody
    Peroxidase Conjugated Secondary Antibody, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nichirei peroxidase conjugated secondary antibody
    Peroxidase Conjugated Secondary Antibody, supplied by Nichirei, used in various techniques. Bioz Stars score: 92/100, based on 419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega peroxidase conjugated secondary antibody
    Peroxidase Conjugated Secondary Antibody, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies peroxidase conjugated secondary antibodies
    Peroxidase Conjugated Secondary Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Nacalai peroxidase conjugated secondary antibodies
    Peroxidase Conjugated Secondary Antibodies, supplied by Nacalai, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche peroxidase conjugated secondary antibodies
    Peroxidase Conjugated Secondary Antibodies, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology peroxidase conjugated secondary antibody
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 5443 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher peroxidase conjugated secondary antibodies
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agrisera peroxidase conjugated secondary antibody
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibody, supplied by Agrisera, used in various techniques. Bioz Stars score: 90/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ICN Pharmaceuticals peroxidase conjugated secondary antibody
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibody, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam secondary peroxidase conjugated antibodies
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Secondary Peroxidase Conjugated Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime peroxidase conjugated secondary antibody
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibody, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 563 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biocare Medical peroxidase conjugated secondary antibodies
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibodies, supplied by Biocare Medical, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim peroxidase conjugated secondary antibodies
    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA <t>antibody,</t> followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a <t>peroxidase-conjugated</t> <t>secondary</t> antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p
    Peroxidase Conjugated Secondary Antibodies, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA antibody, followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p

    Journal: Scientific Reports

    Article Title: Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

    doi: 10.1038/s41598-020-65589-7

    Figure Lengend Snippet: Agonist-induced DOP receptor phosphorylation and internalization. ( A,B top) Stably HA-tagged hDOP receptor-expressing HEK293 cells were preincubated with anti-HA antibody, followed by stimulation with 10 µM DPDPE, DADLE, SNC80, ADL5859, AR-M1000390, deltorphin I, deltorphin II, [Met]-enkephalin, [Leu]-enkephalin, fentanyl, (−)-methadone, morphine, morphine-6-glucuronide (M6G), buprenorphine (BUP), norbuprenorphine (norBUP) or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained, and subsequently examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (A,B bottom) HEK293 cells stably expressing HA-hDOP receptor were stimulated with the compounds listed in (A) or vehicle at concentrations ranging from 10 -9 to 10 -5 M for 10 min at 37 °C. Lysates were immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 3-5. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle or 10 µM of compounds listed in ( A ) for 30 min at 37 °C. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are means ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p

    Article Snippet: Cells were washed several times with PBS and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz; Heidelberg, Germany).

    Techniques: Stable Transfection, Expressing, Staining, Confocal Microscopy, Labeling, Enzyme-linked Immunosorbent Assay

    Time course of DPDPE-induced DOP receptor phosphorylation, concentration-dependent DOP receptor phosphorylation and internalization. ( A ) HEK293 cells stably expressing HA-tagged hDOP receptor were preincubated with anti-HA antibody and stimulated with the indicated DPDPE concentrations for 30 min at 37 °C. After stimulation, cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (B) Cells described in (A) were preincubated with anti-HA antibody and stimulated with the indicated DPDPE concentrations for 30 min at 37 °C. After fixation, cells were labeled with a peroxidase-conjugated secondary antibody and receptor internalization was measured by enzyme-linked immunosorbent assay. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors compared to untreated cells. Data are means ± SEM of four independent experiments performed in quadruplicate. (C,D) Cells described in ( A ) were exposed to 1 µM DPDPE for the indicated times and temperatures ( C ) at 37 °C, ( D ) at 22 °C (room temperature, RT) and lysates were immunoblotted with anti-pT361 or anti-pS363 antibodies. Blots are representative of n = 4 ( C ) or n = 5 ( D ) independent experiments. (E) Cells described in (A) were preincubated with anti-HA antibody and thereafter stimulated with 1 µM DPDPE for the indicated durations at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (F) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with 10 µM DPDPE for the indicated durations at 37 °C. Cells were fixed and labeled with peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are mean ± SEM of five independent experiments performed in quadruplicate.

    Journal: Scientific Reports

    Article Title: Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

    doi: 10.1038/s41598-020-65589-7

    Figure Lengend Snippet: Time course of DPDPE-induced DOP receptor phosphorylation, concentration-dependent DOP receptor phosphorylation and internalization. ( A ) HEK293 cells stably expressing HA-tagged hDOP receptor were preincubated with anti-HA antibody and stimulated with the indicated DPDPE concentrations for 30 min at 37 °C. After stimulation, cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (B) Cells described in (A) were preincubated with anti-HA antibody and stimulated with the indicated DPDPE concentrations for 30 min at 37 °C. After fixation, cells were labeled with a peroxidase-conjugated secondary antibody and receptor internalization was measured by enzyme-linked immunosorbent assay. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors compared to untreated cells. Data are means ± SEM of four independent experiments performed in quadruplicate. (C,D) Cells described in ( A ) were exposed to 1 µM DPDPE for the indicated times and temperatures ( C ) at 37 °C, ( D ) at 22 °C (room temperature, RT) and lysates were immunoblotted with anti-pT361 or anti-pS363 antibodies. Blots are representative of n = 4 ( C ) or n = 5 ( D ) independent experiments. (E) Cells described in (A) were preincubated with anti-HA antibody and thereafter stimulated with 1 µM DPDPE for the indicated durations at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (F) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with 10 µM DPDPE for the indicated durations at 37 °C. Cells were fixed and labeled with peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as the percentage of internalized receptors in agonist-treated cells compared to untreated cells. Data are mean ± SEM of five independent experiments performed in quadruplicate.

    Article Snippet: Cells were washed several times with PBS and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz; Heidelberg, Germany).

    Techniques: Concentration Assay, Stable Transfection, Expressing, Staining, Confocal Microscopy, Labeling, Enzyme-linked Immunosorbent Assay

    Characterization of phosphosite-specific DOP receptor antibodies using λ-phosphatase and receptor mutants. ( A ) Schematic representation of the human DOP receptor (hDOP). Potential intracellular phosphorylation sites are depicted in gray. T361 was targeted for the generation of the phosphosite-specific anti-pT361 antibody and anti-pS363 antibody was acquired commercially. (B) Characterization of phosphosite-specific antibodies directed against T361 and S363 using λ-phosphatase. Stably HA-tagged hDOP-receptor expressing HEK293 cells were either not treated (−) or treated (+) with 10 µM DADLE for 10 min. Lysates were then either not incubated (−) or incubated (+) with λ-phosphatase and immunoblotted with the phosphosite-specific antibodies anti-pT361 {5038} or anti-pS363. Blots were stripped and reprobed with the anti-HA antibody {2238} as a loading control. Blots are representative, n = 3. Molecular mass markers (kDA) are indicated, left. (C) Sequence of the carboxyl-terminal tail of hDOP receptor showing all potential phosphorylation sites. Serine (S) and threonine (T) residues indicated were exchanged to alanine. (D) HEK293 cells stably expressing HA-hDOP receptor, T361A/S363A or 7 S/TA were not treated (−) or treated (+) with 1 µM DPDPE for 10 min and lysates were immunoblotted with the antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 5. (E) Cells described in (D) were preincubated with antibody to HA-tag and subsequently exposed to 10 µM DPDPE or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (F) Receptor internalization was quantified by ELISA. Cells described in (D) were preincubated with antibody to HA-tag and stimulated with 10 µM DPDPE or vehicle at 37 °C for 30 min. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by enzyme-linked immunosorbent assay and quantified as the percentage of internalized receptors in DPDPE-treated cells. Data are mean ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p

    Journal: Scientific Reports

    Article Title: Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

    doi: 10.1038/s41598-020-65589-7

    Figure Lengend Snippet: Characterization of phosphosite-specific DOP receptor antibodies using λ-phosphatase and receptor mutants. ( A ) Schematic representation of the human DOP receptor (hDOP). Potential intracellular phosphorylation sites are depicted in gray. T361 was targeted for the generation of the phosphosite-specific anti-pT361 antibody and anti-pS363 antibody was acquired commercially. (B) Characterization of phosphosite-specific antibodies directed against T361 and S363 using λ-phosphatase. Stably HA-tagged hDOP-receptor expressing HEK293 cells were either not treated (−) or treated (+) with 10 µM DADLE for 10 min. Lysates were then either not incubated (−) or incubated (+) with λ-phosphatase and immunoblotted with the phosphosite-specific antibodies anti-pT361 {5038} or anti-pS363. Blots were stripped and reprobed with the anti-HA antibody {2238} as a loading control. Blots are representative, n = 3. Molecular mass markers (kDA) are indicated, left. (C) Sequence of the carboxyl-terminal tail of hDOP receptor showing all potential phosphorylation sites. Serine (S) and threonine (T) residues indicated were exchanged to alanine. (D) HEK293 cells stably expressing HA-hDOP receptor, T361A/S363A or 7 S/TA were not treated (−) or treated (+) with 1 µM DPDPE for 10 min and lysates were immunoblotted with the antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 5. (E) Cells described in (D) were preincubated with antibody to HA-tag and subsequently exposed to 10 µM DPDPE or vehicle for 30 min at 37 °C. Cells were fixed, permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative from one of three independent experiments. Scale bar, 20 µm. (F) Receptor internalization was quantified by ELISA. Cells described in (D) were preincubated with antibody to HA-tag and stimulated with 10 µM DPDPE or vehicle at 37 °C for 30 min. Cells were fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by enzyme-linked immunosorbent assay and quantified as the percentage of internalized receptors in DPDPE-treated cells. Data are mean ± SEM of five independent experiments performed in quadruplicate. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test (*p

    Article Snippet: Cells were washed several times with PBS and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz; Heidelberg, Germany).

    Techniques: Stable Transfection, Expressing, Incubation, Sequencing, Staining, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Labeling

    Antagonist-selective inhibition of DPDPE-induced DOP receptor phosphorylation, internalization and G protein signaling. ( A ) Stably HA-hDOP receptor-expressing HEK293 cells were either not preincubated (−) or preincubated (+) with 5 µM naloxone, naltrindole, naltriben or naltrexone for 30 min at 37 °C, then stimulated with vehicle (water, -) or with 1 µM DPDPE (+) for 10 min at 37 °C. Cell lysates were then immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 4. (B) Cells described in ( A ) were preincubated with anti-HA antibody and then treated with vehicle (DMSO), 5 µM naloxone, naltrindole or naltriben and with or without 1 µM DPDPE for 10 min at 37 °C. After fixation, cells were permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle (DMSO), 5 µM naloxone, naltrindole or naltriben and with or without 1 µM DPDPE for 10 min at 37 °C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from five independent experiments performed in quadruplicate. *p

    Journal: Scientific Reports

    Article Title: Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

    doi: 10.1038/s41598-020-65589-7

    Figure Lengend Snippet: Antagonist-selective inhibition of DPDPE-induced DOP receptor phosphorylation, internalization and G protein signaling. ( A ) Stably HA-hDOP receptor-expressing HEK293 cells were either not preincubated (−) or preincubated (+) with 5 µM naloxone, naltrindole, naltriben or naltrexone for 30 min at 37 °C, then stimulated with vehicle (water, -) or with 1 µM DPDPE (+) for 10 min at 37 °C. Cell lysates were then immunoblotted with antibodies to pT361 or pS363. Blots were stripped and reprobed with the anti-HA antibody. Blots are representative, n = 4. (B) Cells described in ( A ) were preincubated with anti-HA antibody and then treated with vehicle (DMSO), 5 µM naloxone, naltrindole or naltriben and with or without 1 µM DPDPE for 10 min at 37 °C. After fixation, cells were permeabilized, immunofluorescently stained and examined using confocal microscopy. Images are representative, n = 3. Scale bar, 20 µm. (C) Cells described in ( A ) were preincubated with anti-HA antibody and stimulated with vehicle (DMSO), 5 µM naloxone, naltrindole or naltriben and with or without 1 µM DPDPE for 10 min at 37 °C. Cells were then fixed and labeled with a peroxidase-conjugated secondary antibody. Receptor internalization was measured by ELISA and quantified as percentage of internalized receptors in agonist-treated cells. Data are means ± SEM from five independent experiments performed in quadruplicate. *p

    Article Snippet: Cells were washed several times with PBS and incubated with a peroxidase-conjugated secondary antibody (Santa Cruz; Heidelberg, Germany).

    Techniques: Inhibition, Stable Transfection, Expressing, Staining, Confocal Microscopy, Labeling, Enzyme-linked Immunosorbent Assay