hrp-conjugated secondary antibodies Search Results


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  • 99
    Boster Bio goat anti rabbit igg h l secondary antibody hrp conjugate
    Goat Anti Rabbit Igg H L Secondary Antibody Hrp Conjugate, supplied by Boster Bio, used in various techniques. Bioz Stars score: 99/100, based on 1006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1006 article reviews
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    Thermo Fisher hrp conjugated secondary ab
    Hrp Conjugated Secondary Ab, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology hrp conjugated secondary antibodies
    siRNA directed toward the <t>uPA</t> promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by <t>HRP-conjugated</t> secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P
    Hrp Conjugated Secondary Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 4960 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated secondary antibodies/product/Santa Cruz Biotechnology
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    hrp conjugated secondary antibodies - by Bioz Stars, 2020-12
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    Thermo Fisher horseradish peroxidase conjugated secondary antibodies
    siRNA directed toward the <t>uPA</t> promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by <t>HRP-conjugated</t> secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P
    Horseradish Peroxidase Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5537 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc hrp conjugated secondary antibody
    siRNA directed toward the <t>uPA</t> promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by <t>HRP-conjugated</t> secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P
    Hrp Conjugated Secondary Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated secondary antibody/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2135 article reviews
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    Bio-Rad hrp conjugated secondary antibody
    siRNA directed toward the <t>uPA</t> promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by <t>HRP-conjugated</t> secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P
    Hrp Conjugated Secondary Antibody, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrp conjugated secondary antibody/product/Bio-Rad
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    hrp conjugated secondary antibody - by Bioz Stars, 2020-12
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    94
    Agilent technologies hrp conjugated secondary antibodies
    Binding of recombinant <t>CD2831</t> full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by <t>HRP-conjugated-secondary</t> antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.
    Hrp Conjugated Secondary Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 2698 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology horseradish peroxidase hrp conjugated secondary antibody
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp Conjugated Secondary Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher horseradish peroxidase hrp conjugated secondary antibodies
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam hrp conjugated secondary antibodies
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Hrp Conjugated Secondary Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore horseradish peroxidase hrp conjugated secondary antibodies
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp Conjugated Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad horseradish peroxidase hrp conjugated secondary antibody
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Horseradish Peroxidase Hrp Conjugated Secondary Antibody, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Beyotime hrp conjugated secondary antibodies
    PepE target the <t>TrxR1</t> through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. <t>HRP-conjugated</t> streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P
    Hrp Conjugated Secondary Antibodies, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies horseradish peroxidase hrp conjugated secondary antibodies
    Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. <t>Streptavidin</t> reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with <t>streptavidin-HRP.</t> Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.
    Horseradish Peroxidase Hrp Conjugated Secondary Antibodies, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 533 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam horseradish peroxidase hrp conjugated secondary antibodies
    Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. <t>Streptavidin</t> reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with <t>streptavidin-HRP.</t> Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.
    Horseradish Peroxidase Hrp Conjugated Secondary Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega horseradish peroxidise conjugated secondary antibodies
    Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish <t>peroxidise-conjugated</t> secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P
    Horseradish Peroxidise Conjugated Secondary Antibodies, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igm heavy chain secondary antibody
    Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish <t>peroxidise-conjugated</t> secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P
    Goat Anti Mouse Igm Heavy Chain Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory hrp conjugated secondary antibodies
    Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with <t>streptavidin-HRP</t> to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value
    Hrp Conjugated Secondary Antibodies, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 93/100, based on 448 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime horseradish peroxidase hrp conjugated secondary antibodies
    Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with <t>streptavidin-HRP</t> to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value
    Horseradish Peroxidase Hrp Conjugated Secondary Antibodies, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HRP Conjugated Goat Anti human IgM u chain specific secondary antibody This HRP conjugated antibody is specific for human IgM and shows no cross reactivity with human IgA IgG
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    This antibody is purified from antiserum by immunoaffinity chromatography which removes essentially all rabbit serum proteins except the specific antibody for rat IgG
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    Image Search Results


    siRNA directed toward the uPA promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by HRP-conjugated secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells 1

    doi:

    Figure Lengend Snippet: siRNA directed toward the uPA promoter prevents radiation-induced hypomethylation. (A) Schematic depiction of the uPA promoter showing shRNA target region, Sp1 binding sites, and the region selected for MSP. (B) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, bisulfite-treated genomic DNA was subjected to MSP and followed by agarose gel analysis. (C) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, conditioned media were subjected to fibrin zymography, total RNA was used to perform RT-PCR, and cell lysates were used for immunoblot analysis as indicated. (D) The band intensities were quantified and represented as arbitrary units. (E) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were fixed and subjected to immunocytochemistry with anti-uPA antibody (1:200) followed by HRP-conjugated secondary antibody (1:500). The slides were treated with DAB, counterstained with hematoxylin, and observed under x25 objective. Representative images of each treatment group are shown. (F) IOMM-Lee and SF3061 (1 x 10 5 ) cells were transfected with pSV or puPA for 36 hours before irradiation. After overnight incubation, cells were transferred to transwell chambers coated with matrigel (1 mg/ml) and allowed to invade for 24 hours. Percentage of invaded cells in each treatment group was calculated and plotted. Each blot/image is representative of three independent experiments, and each column represents a mean of three values (asterisk represents significant value of P

    Article Snippet: DNMT1, phospho extracellular signal-regulated kinase (pERK), ERK, MEK, phospho MAP kinase kinase (pMEK), cRaf, pcRAF, uPA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: shRNA, Binding Assay, Transfection, Irradiation, Incubation, Agarose Gel Electrophoresis, Zymography, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry

    Radiation treatment induces hypomethylation in vivo . Nude mice were intracranially implanted with IOMM-Lee cells as described in the Materials and Methods section. (A) Formalin-fixed brain sections were subjected to H E staining and observed under x20 objective. Each image represents a brain section of five animals. (B) Total RNA was extracted from frozen brain tissues, and RT-PCR was performed for uPA and GAPDH. Tissue lysates were analyzed for uPA expression by Western blot analysis. (C) The band intensities of RT-PCR gels and Western blots were quantified and represented as arbitrary units. (D) Genomic DNA from frozen brain tissues was extracted, treated with bisulfite, and subjected to MSP targeting the uPA promoter (MP, methylated primer; UMP, unmethylated primer). (E) The band intensities were quantified and represented as arbitrary units. (F) Immunostaining with anti-DNMT1 antibody and non-specific IgG (NsIgG) was performed on formalin-fixed brain sections from animals implanted with IOMM-Lee cells followed by treatment with HRP-conjugated secondary antibody and DAB staining. Representative photomicrographs (x200) are shown in comparison to normal mouse brain sections. (G) Immunohistochemical analysis for DNMT1 was performed on tissue microarrays and clinical meningioma samples. Representative images (x400) are shown. Each column represents a mean value of brain tissues from three different animals (* P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells 1

    doi:

    Figure Lengend Snippet: Radiation treatment induces hypomethylation in vivo . Nude mice were intracranially implanted with IOMM-Lee cells as described in the Materials and Methods section. (A) Formalin-fixed brain sections were subjected to H E staining and observed under x20 objective. Each image represents a brain section of five animals. (B) Total RNA was extracted from frozen brain tissues, and RT-PCR was performed for uPA and GAPDH. Tissue lysates were analyzed for uPA expression by Western blot analysis. (C) The band intensities of RT-PCR gels and Western blots were quantified and represented as arbitrary units. (D) Genomic DNA from frozen brain tissues was extracted, treated with bisulfite, and subjected to MSP targeting the uPA promoter (MP, methylated primer; UMP, unmethylated primer). (E) The band intensities were quantified and represented as arbitrary units. (F) Immunostaining with anti-DNMT1 antibody and non-specific IgG (NsIgG) was performed on formalin-fixed brain sections from animals implanted with IOMM-Lee cells followed by treatment with HRP-conjugated secondary antibody and DAB staining. Representative photomicrographs (x200) are shown in comparison to normal mouse brain sections. (G) Immunohistochemical analysis for DNMT1 was performed on tissue microarrays and clinical meningioma samples. Representative images (x400) are shown. Each column represents a mean value of brain tissues from three different animals (* P

    Article Snippet: DNMT1, phospho extracellular signal-regulated kinase (pERK), ERK, MEK, phospho MAP kinase kinase (pMEK), cRaf, pcRAF, uPA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: In Vivo, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Methylation, Immunostaining, Immunohistochemistry

    Binding of recombinant CD2831 full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by HRP-conjugated-secondary antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.

    Journal: Scientific Reports

    Article Title: Dual role of the colonization factor CD2831 in Clostridium difficile pathogenesis

    doi: 10.1038/s41598-019-42000-8

    Figure Lengend Snippet: Binding of recombinant CD2831 full-length protein and subdomains to collagen. ( A ) Schematic representation of CD2831 domains organization displays the Leader Peptide at the N-terminus (orange) followed by a Short Repeat Region (yellow) in which repeated amino acids sequences are marked in bold, two collagen-binding domains (blue) and a Proline-rich region (green) recognized by the protease ZmpI/PPEP-1 that cleaves within the seven NPP amino acids sequences of the domain. The charged tail and the hydrophobic domain are present at the C-terminus (red). The protein is cleaved between the threonyl and glycil residues of the PPKTG motif and transferred to the cell-wall peptidoglycan by the Sortase B enzyme. Blue, black and grey lines indicate the length of recombinant CD2831 subdomains compared to the full-length. ( B ) Immunofluorescence of 1 µM of CD2831 (green) binding to collagen (red) produced by IMR90 cells. Collagen staining results from a mix of antibodies against collagen types I, III and V in a 1:1:1 ratio. Binding of the proteins was detected by using rabbit anti-CD2831 serum and secondary Alexa Fluor-conjugated antibodies. ( C ) Human fibroblasts IMR90 were cultured into collagen I-coated 96 well plates for three days after seeding. Cells were then incubated with serially diluted recombinant CD2831 proteins ranging from 0.08 to 10 µM. Binding of CD2831 was detected as above and quantified with a microplate fluorescence reader. ( D ) ELISA plates were coated with 10 μg/ml of purified collagens and incubated with serially diluted recombinant CD2831 proteins ranging from 15 nM to 2 µM. Binding of the proteins was detected using rabbit anti-CD2831 serum, followed by HRP-conjugated-secondary antibody. CWP25 protein was used as negative control. CD2831 CBD1 and CD2831 CBD2 represent the previously described subdomains 1 and 2.

    Article Snippet: After blocking with PBS containing 0.05% Tween-20 (PBS-T) and 10% (w/v) milk (Sigma, USA), proteins were detected with rabbit anti-CD2831 serum diluted 1:1000 for 1 hour at RT, followed by the HRP-conjugated secondary antibodies (Dako, Denmark) diluted 1:2000 for 1 hour at RT.

    Techniques: Binding Assay, Recombinant, Immunofluorescence, Produced, Staining, Cell Culture, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay, Purification, Negative Control

    PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P

    Journal: Redox Biology

    Article Title: Peperomin E and its orally bioavailable analog induce oxidative stress-mediated apoptosis of acute myeloid leukemia progenitor cells by targeting thioredoxin reductase

    doi: 10.1016/j.redox.2019.101153

    Figure Lengend Snippet: PepE target the TrxR1 through binding its Sec residue. (A) Inhibition assay of purified recombinant human TrxR1 or TrxR1 without Sec498 residue (nSec498, 0.1 μM) by PepE. The NADPH-reduced proteins were incubated with the indicated concentrations of PepE, and the enzyme's activity was determined by the DTNB assay (Data are presented as mean ± SD, n = 6). (B) Molecular docking of PepE with TrxR1 was carried out using the covalent docking protocol in the Schrodinger Suite. The yellow line indicated the covalent carbon bond, the green dotted lines indicate the hydrogen bonds. (C) Comparison of the binding activity between PepE and TrxR1 protein (red line), PepE and TrxR1 nSec498 protein (blue line), and PepA and TrxR1 protein (black line) through BLI analysis. (D) Kinetic analysis of the interaction between NADPH reduced TrxR1 (0.9 μM) or NADPH reduced TrxR1 nSec498 (0.9 μM) protein and PepE by BLI. The Super Streptavidin (SSA) biosensor tips coated with proteins (0.9 μM) were dipped in increasing concentrations of PepE (0.625, 1.25, 2.5, 5 and 10 μM) to measure the binding affinity of PepE to the proteins (K on ) and subsequently moved to wells containing buffer to measure dissociation rates (K dis ). The affinity constant (K D ) was calculated as the ratio of the K dis to the K on. (E) Kinetic analysis of the interaction between TrxR1 (0.9 μM) and different concentrations of PepA (1.25, 2.5, 5, 10 and 20 μM) by BLI. (F) The interaction of the PepE with the Sec residue in the C -terminal active center of TrxR1. HRP-conjugated streptavidin (HRP-Strep) detection of BIAM labeling of free Selenol in TrxR1 enzyme at pH 6.5 after this enzyme was incubated with different concentrations of PepE (1, 5 and 10 μM), DMSO(negative control), and DNCB (positive control, 5 μM), ** P

    Article Snippet: Horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit), human TrxR1 shRNA plasmids, control shRNA plasmids, TrxR1 CRISPR activation plasmids, control CRISPR activation plasmids were obtained from Santa Cruz Biotech.

    Techniques: Binding Assay, Size-exclusion Chromatography, Inhibition, Purification, Recombinant, Incubation, Activity Assay, DTNB Assay, Labeling, Positive Control

    Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.

    Journal: PLoS ONE

    Article Title: BioID identifies proteins involved in the cell biology of caveolae

    doi: 10.1371/journal.pone.0209856

    Figure Lengend Snippet: Expression of cavin1-myc-BirA* as a tool to label caveolar proteins. A . Schematic representation of constructs used in this study. B . Expression of constructs used in this study, analysed by Western blotting with anti-myc antibodies. C . Distribution of biotin in transfected cells, compared with caveolae labelled with antibodies against caveolin1. Anti-myc antibodies reveal the location of the indicated BirA* construct. Streptavidin reveals the location of biotinylated proteins. Arrowheads highlight examples of streptavidin-stained caveolae. Bar is 20 microns. Single confocal sections acquired with 63x objective. D . Blot of biotinylated proteins labelled with streptavidin-HRP. Cells were transfected with the myc-BirA* construct indicated at the top of each lane on the blot, and incubated with exogenous biotin before solubilisation. The band labelled 1 in the cavin1-myc-BirA* lane is the correct size to be cavin1-myc-BirA*, 2 is the correct size to be endogenous cavin1, and 3 is the correct size to be caveolin1. E . Western blot labelled with caveolin1 antibody. Cells were transfected with cavin1-myc-BirA* and incubated with exogenous biotin for the times shown, before solubilisation and precipitation of biotinylated proteins with immobilised streptavidin.

    Article Snippet: Horseradish peroxidase (HRP)-conjugated secondary antibodies were from DAKO and Streptavidin HRP from Cell Signalling (Cat# 3999).

    Techniques: Expressing, Construct, Western Blot, Transfection, Staining, Incubation

    Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish peroxidise-conjugated secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P

    Journal: Scientific Reports

    Article Title: Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo

    doi: 10.1038/s41598-017-07494-0

    Figure Lengend Snippet: Characterisation and purification of the royal jelly (RJ) component responsible for elevated MMP-9 production. ( A ) Heat and proteinase K treatment was performed by incubation of water royal jelly extract (WRJE) at 100 °C for 5 min and incubation with 150 μg/ml proteinase K for 1 h at 40 °C followed by heating to 98 °C for 10 min to inactivate the enzyme. Treated WRJE was incubated with HaCaT cells and conditioned equal volumes of the culture media were collected and subjected to gelatine zymography. Densitometric quantification of MMP-9 activity in culture media is presented. ( B ) Heat-treated WRJE was fractionated by a reverse phase-high performance liquid chromatography (RP-HPLC) on a C18 column (250 × 4.6 mm, 5 μm) at a flow rate 0.3 ml/min, with elution using a 10–90% gradient of acetonitrile (containing 0.1% (v/v) trifluoroacetic acid) for 85 min. ( C ) The HPLC fractions were assayed for MMP-9 induction. ( D ) HPLC fractions with maximal MMP-9 activity (51 to 59 min) were used for identification of MMP-9 inducer and were subjected to 16.5% Tricine-SDS-PAGE gels. Defensin-1 (5.5 kDa) was detected by Western blotting using a rabbit polyclonal anti-honeybee defensin-1 antibody diluted 1:2000 in blocking buffer. Horseradish peroxidise-conjugated secondary antibodies were applied. (The gels/blots with indicated cropping lines are shown in Supplementary Fig. S3 ). White line in gel indicates the place where two gels were spliced together. Data are expressed as means and SEMs of three independent measurements. Asterisks indicate a significant difference from the untreated group, * P

    Article Snippet: Rabbit polyclonal anti-bee defensin-1 (Def-1) was purchased from GenCust Europe (Luxembourg), and horseradish peroxidise-conjugated secondary antibodies were obtained from Promega (USA).

    Techniques: Purification, Incubation, Zymography, Activity Assay, High Performance Liquid Chromatography, Flow Cytometry, SDS Page, Western Blot, Blocking Assay

    Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with streptavidin-HRP to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value

    Journal: PLoS ONE

    Article Title: Degradation of Internalized ?v?5 Integrin Is Controlled by uPAR Bound uPA: Effect on ?1 Integrin Activity and ?-SMA Stress Fiber Assembly

    doi: 10.1371/journal.pone.0033915

    Figure Lengend Snippet: Integrin β5 protein expression is increased on TGFβ-induced Mfs. HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with streptavidin-HRP to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value

    Article Snippet: HRP-conjugated anti-Streptavidin antibody and all HRP-conjugated secondary antibodies were from Jackson Laboratories.

    Techniques: Expressing, Flow Cytometry, Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Real-time Polymerase Chain Reaction