rnasin Thermo Fisher Search Results


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  • 90
    Thermo Fisher rnasin
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of <t>RNasin</t> or <t>RNase</t> A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
    Rnasin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3892 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribolock rnase inhibitor thermo fisher scientific
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of <t>RNasin</t> or <t>RNase</t> A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
    Ribolock Rnase Inhibitor Thermo Fisher Scientific, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega rnasin
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of <t>RNasin</t> or <t>RNase</t> A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
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    Thermo Fisher ribolock rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
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    Thermo Fisher super rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Super Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase inhibitor superase in
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
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    Thermo Fisher riboblock rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Riboblock Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaseout rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Rnaseout Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribolocktm rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Ribolocktm Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superasin rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Superasin Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher multiscribe rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Multiscribe Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher geneamp rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Geneamp Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher prime rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Prime Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnasin inhibitors
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Rnasin Inhibitors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase inhibitor anti rnase
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Rnase Inhibitor Anti Rnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ribolock rnase inhibitor 20u
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
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    Thermo Fisher ribolock rnase inhibitor kits
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
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    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Thermo Fisher rnaseout recombinant rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Rnaseout Recombinant Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher recombinant human rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Thermo Fisher rnasine
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Thermo Fisher rnase inhibitior
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Thermo Fisher multscribe reverse transcriptase rnase inhibitor mix
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Thermo Fisher recombinant human placenta rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    Interactions between PIWI proteins and histone modification-related proteins. ( A ) <t>Co-immunoprecipitation</t> (Co-IP) assay of PIWIL1 and WDR5 by anti-HA agarose beads followed by immunoblots with indicated antibodies (middle). ( B ) Co-IP assay of PIWIL4 and WDR5. ( C ) Co-IP assay of PIWIL1 and PIWIL4. ( D ) Co-IP assay of PIWIL1 and WDR5 after <t>RNase</t> treatment by anti-HA agarose beads followed by immunoblots. ( E ) Co-IP assay of PIWIL1 and WDR5 in the presence or absence of MLL3 or UTX by anti-HA antibodies followed by immunoblots. ( F ) Co-IP assay of PIWIL1 and MLL3 in the presence or absence of WDR5 or UTX. ( G ) Co-IP assay of PIWIL1 and UTX in the presence or absence of WDR5 or MLL3. ( H ) Endogenous co-IP assay of PIWIL1 and WDR5 in MCF7 cells by using anti-PIWIL1 antibody. ( I ) association of WDR5 with the TRAIL promoter under the overexpression of pi-sno75 was detected by ChIP-qPCR. The relative enrichment was calculated by normalizing the quantity of TRAIL DNA against the quantity of input. ( J ) The association of PIWIL1 with the TRAIL promoter in the absence of WDR5 was detected by ChIP-qPCR. ( K ) The association of WDR5 with the TRAIL promoter in the absence of PIWIL1 was detected by ChIP-qPCR. Bar graphs represent mean ± SD ( n = 3). *, statistically significant, ≤0.05.
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    Thermo Fisher human rnase inhibitors
    Interactions between PIWI proteins and histone modification-related proteins. ( A ) <t>Co-immunoprecipitation</t> (Co-IP) assay of PIWIL1 and WDR5 by anti-HA agarose beads followed by immunoblots with indicated antibodies (middle). ( B ) Co-IP assay of PIWIL4 and WDR5. ( C ) Co-IP assay of PIWIL1 and PIWIL4. ( D ) Co-IP assay of PIWIL1 and WDR5 after <t>RNase</t> treatment by anti-HA agarose beads followed by immunoblots. ( E ) Co-IP assay of PIWIL1 and WDR5 in the presence or absence of MLL3 or UTX by anti-HA antibodies followed by immunoblots. ( F ) Co-IP assay of PIWIL1 and MLL3 in the presence or absence of WDR5 or UTX. ( G ) Co-IP assay of PIWIL1 and UTX in the presence or absence of WDR5 or MLL3. ( H ) Endogenous co-IP assay of PIWIL1 and WDR5 in MCF7 cells by using anti-PIWIL1 antibody. ( I ) association of WDR5 with the TRAIL promoter under the overexpression of pi-sno75 was detected by ChIP-qPCR. The relative enrichment was calculated by normalizing the quantity of TRAIL DNA against the quantity of input. ( J ) The association of PIWIL1 with the TRAIL promoter in the absence of WDR5 was detected by ChIP-qPCR. ( K ) The association of WDR5 with the TRAIL promoter in the absence of PIWIL1 was detected by ChIP-qPCR. Bar graphs represent mean ± SD ( n = 3). *, statistically significant, ≤0.05.
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    Image Search Results


    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Journal: EMBO Reports

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

    doi: 10.1038/embor.2009.179

    Figure Lengend Snippet: VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Article Snippet: For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Immunofluorescence, Microscopy, Polyacrylamide Gel Electrophoresis

    RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Concentration Assay, Incubation, Blocking Assay, Activity Assay

    RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Activity Assay

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Interactions between PIWI proteins and histone modification-related proteins. ( A ) Co-immunoprecipitation (Co-IP) assay of PIWIL1 and WDR5 by anti-HA agarose beads followed by immunoblots with indicated antibodies (middle). ( B ) Co-IP assay of PIWIL4 and WDR5. ( C ) Co-IP assay of PIWIL1 and PIWIL4. ( D ) Co-IP assay of PIWIL1 and WDR5 after RNase treatment by anti-HA agarose beads followed by immunoblots. ( E ) Co-IP assay of PIWIL1 and WDR5 in the presence or absence of MLL3 or UTX by anti-HA antibodies followed by immunoblots. ( F ) Co-IP assay of PIWIL1 and MLL3 in the presence or absence of WDR5 or UTX. ( G ) Co-IP assay of PIWIL1 and UTX in the presence or absence of WDR5 or MLL3. ( H ) Endogenous co-IP assay of PIWIL1 and WDR5 in MCF7 cells by using anti-PIWIL1 antibody. ( I ) association of WDR5 with the TRAIL promoter under the overexpression of pi-sno75 was detected by ChIP-qPCR. The relative enrichment was calculated by normalizing the quantity of TRAIL DNA against the quantity of input. ( J ) The association of PIWIL1 with the TRAIL promoter in the absence of WDR5 was detected by ChIP-qPCR. ( K ) The association of WDR5 with the TRAIL promoter in the absence of PIWIL1 was detected by ChIP-qPCR. Bar graphs represent mean ± SD ( n = 3). *, statistically significant, ≤0.05.

    Journal: Nucleic Acids Research

    Article Title: An Lnc RNA (GAS5)/SnoRNA-derived piRNA induces activation of TRAIL gene by site-specifically recruiting MLL/COMPASS-like complexes

    doi: 10.1093/nar/gkv214

    Figure Lengend Snippet: Interactions between PIWI proteins and histone modification-related proteins. ( A ) Co-immunoprecipitation (Co-IP) assay of PIWIL1 and WDR5 by anti-HA agarose beads followed by immunoblots with indicated antibodies (middle). ( B ) Co-IP assay of PIWIL4 and WDR5. ( C ) Co-IP assay of PIWIL1 and PIWIL4. ( D ) Co-IP assay of PIWIL1 and WDR5 after RNase treatment by anti-HA agarose beads followed by immunoblots. ( E ) Co-IP assay of PIWIL1 and WDR5 in the presence or absence of MLL3 or UTX by anti-HA antibodies followed by immunoblots. ( F ) Co-IP assay of PIWIL1 and MLL3 in the presence or absence of WDR5 or UTX. ( G ) Co-IP assay of PIWIL1 and UTX in the presence or absence of WDR5 or MLL3. ( H ) Endogenous co-IP assay of PIWIL1 and WDR5 in MCF7 cells by using anti-PIWIL1 antibody. ( I ) association of WDR5 with the TRAIL promoter under the overexpression of pi-sno75 was detected by ChIP-qPCR. The relative enrichment was calculated by normalizing the quantity of TRAIL DNA against the quantity of input. ( J ) The association of PIWIL1 with the TRAIL promoter in the absence of WDR5 was detected by ChIP-qPCR. ( K ) The association of WDR5 with the TRAIL promoter in the absence of PIWIL1 was detected by ChIP-qPCR. Bar graphs represent mean ± SD ( n = 3). *, statistically significant, ≤0.05.

    Article Snippet: RNA immunoprecipitation To detect whether the small RNAs were associated with the PIWI proteins, PBMCs were homogenized using immunoprecipitation (IP) lysis buffer plus RNase inhibitor RNaseOUT (40 units/ml, Invitrogen).

    Techniques: Modification, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction