biotinylated secondary Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98
    Vector Laboratories avidin biotin peroxidase immunocytochemistry biotinylated secondary antibodies
    Avidin Biotin Peroxidase Immunocytochemistry Biotinylated Secondary Antibodies, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase immunocytochemistry biotinylated secondary antibodies/product/Vector Laboratories
    Average 98 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase immunocytochemistry biotinylated secondary antibodies - by Bioz Stars, 2020-08
    98/100 stars
      Buy from Supplier

    90
    Thermo Fisher avidin biotin hrp complex
    Avidin Biotin Hrp Complex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin hrp complex/product/Thermo Fisher
    Average 90 stars, based on 121 article reviews
    Price from $9.99 to $1999.99
    avidin biotin hrp complex - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    99
    Vector Laboratories biotinylated secondary antibody
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Biotinylated Secondary Antibody, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 19271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated secondary antibody/product/Vector Laboratories
    Average 99 stars, based on 19271 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary antibody - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    Agilent technologies avidin biotin system
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Avidin Biotin System, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin system/product/Agilent technologies
    Average 90 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    avidin biotin system - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    99
    Vector Laboratories avidin biotin
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Avidin Biotin, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin/product/Vector Laboratories
    Average 99 stars, based on 981 article reviews
    Price from $9.99 to $1999.99
    avidin biotin - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    The Jackson Laboratory biotinylated secondary
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Biotinylated Secondary, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated secondary/product/The Jackson Laboratory
    Average 90 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    94
    Abcam biotinylated antibody
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Biotinylated Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated antibody/product/Abcam
    Average 94 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    biotinylated antibody - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    90
    Agilent technologies avidin biotin peroxidase linked secondary antibody
    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense <t>biotinylated</t> probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.
    Avidin Biotin Peroxidase Linked Secondary Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase linked secondary antibody/product/Agilent technologies
    Average 90 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase linked secondary antibody - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    92
    Jackson Immuno biotinylated secondary
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Biotinylated Secondary, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 92/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated secondary/product/Jackson Immuno
    Average 92 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    91
    Abcam avidin biotin complex kit
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Complex Kit, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin complex kit/product/Abcam
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    avidin biotin complex kit - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    91
    Thermo Fisher avidin biotin complex reagent
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Complex Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin complex reagent/product/Thermo Fisher
    Average 91 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    avidin biotin complex reagent - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    88
    Boster Bio strept avidin biotin complex
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Strept Avidin Biotin Complex, supplied by Boster Bio, used in various techniques. Bioz Stars score: 88/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strept avidin biotin complex/product/Boster Bio
    Average 88 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
    strept avidin biotin complex - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    99
    Vector Laboratories avidin biotin technique
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Technique, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin technique/product/Vector Laboratories
    Average 99 stars, based on 196 article reviews
    Price from $9.99 to $1999.99
    avidin biotin technique - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Millipore avidin biotin conjugated secondary antibody
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Conjugated Secondary Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin conjugated secondary antibody/product/Millipore
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    avidin biotin conjugated secondary antibody - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Vector Laboratories avidin biotin peroxidase
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Peroxidase, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 1879 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase/product/Vector Laboratories
    Average 99 stars, based on 1879 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    92
    Agilent technologies avidin biotin peroxidase complex
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Peroxidase Complex, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 1212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase complex/product/Agilent technologies
    Average 92 stars, based on 1212 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase complex - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    88
    Agilent technologies avidin biotin peroxidase amplification
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Peroxidase Amplification, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase amplification/product/Agilent technologies
    Average 88 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase amplification - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    99
    Abcam avidin biotin peroxidase
    A summary diagram illustrating <t>biotinylated</t> dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).
    Avidin Biotin Peroxidase, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase/product/Abcam
    Average 99 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    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: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin peroxidase conjugated secondary antibody/product/Millipore
    Average 99 stars, based on 56 article reviews
    Price from $9.99 to $1999.99
    avidin biotin peroxidase conjugated secondary antibody - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    89
    Agilent technologies avidin biotin complex abc kit
    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 Complex Abc Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 89/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/avidin biotin complex abc kit/product/Agilent technologies
    Average 89 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    avidin biotin complex abc kit - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    Image Search Results


    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

    Journal: Nature cell biology

    Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

    doi: 10.1038/ncb3595

    Figure Lengend Snippet: PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

    Article Snippet: Sections were incubated overnight in a humid chamber at 4°C with antibody against Ki-67 purchased from Cell signaling technology company (Clone [D2H10], Cat. #9027, 1:1600 dilution) followed by biotinylated secondary antibody (Vector laboratories) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (Dako), and sections were counterstained with hematoxylin before mounting.

    Techniques: Fractionation, Reverse Transcription Polymerase Chain Reaction, Expressing, Confocal Microscopy, Imaging, Cotransfection, Construct, Activation Assay, Quantitative RT-PCR, In Silico, Binding Assay, Incubation, In Vitro, Labeling, Transfection, Concentration Assay, Over Expression

    A summary diagram illustrating biotinylated dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).

    Journal: The Journal of Neuroscience

    Article Title: Innervation of Histaminergic Tuberomammillary Neurons by GABAergic and Galaninergic Neurons in the Ventrolateral Preoptic Nucleus of the Rat

    doi: 10.1523/JNEUROSCI.18-12-04705.1998

    Figure Lengend Snippet: A summary diagram illustrating biotinylated dextran injection sites at six levels of the preoptic area. The VLPO is most prominent in schematics c , c′ , d , and d′ (in light gray ). Asterisks denote cases VLPO 11, VLPO 38, and R 1059 in which biotinylated dextran injections were predominantly located within the VLPO (see Results).

    Article Snippet: Sections were incubated at room temperature in primary antiserum overnight (12–16 hr) and in biotinylated secondary antiserum (Jackson ImmunoResearch, West Grove, PA; donkey, 1:1000) for 1 hr.

    Techniques: Injection

    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