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Promega u reaction rnasin
U Reaction Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Position-specific effect of ribonucleotides on the cleavage activity of human topoisomerase II
Article Snippet: To examine topoisomerase II-mediated cleavage of the 5′-end-labeled RNA/DNA hybrid, cleavage reactions were performed as described above, but in the presence of 40 U/reaction RNasin (Promega) to inhibit RNase reactions.

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  • 99
    Promega rnase inhibitor
    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative <t>RNase</t> protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total <t>RNA</t> from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).
    Rnase Inhibitor, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase inhibitor/product/Promega
    Average 99 stars, based on 1229 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-05
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    92
    Promega np 40 buffer
    SLBP is bound to histone mRNAs in the cell. ( A ) Polyribosomes isolated from mouse myeloma cells were suspended in buffer containing 10 mM EDTA as described in Materials and Methods. α-SLBP was added in the presence of either 0.1% Tween-20 (lanes 3 and 4) or 0.1% <t>NP-40</t> (lanes 5 and 6). RNA was prepared from the immunoprecipitates (lanes 3 and 5) and from the supernatant fraction (lanes 4 and 6) and analyzed by S1 nuclease mapping using the histone H3-614 gene as a probe. RNA prepared from polyribosomes was analyzed in lane 2 and yeast tRNA in lane 1. There is a small size differences (2–4 nt) observed between the total RNA (lane 2) and the immunoprecipitated mRNA compared with the RNA that did not immunoprecipitate. The final lane is pUC18 digested with HpaII. ( B ) Immunoprecipitations were performed from mouse myeloma polyribosomes with preimmune serum (pi) (lanes 4 and 9), α-SLBP (lanes 5 and 10), α-SLBP preincubated with 1 μg of antigenic peptide (lanes 6 and 11) or with an unrelated antibody, α-CUL (lanes 7 and 12). RNA was prepared from both supernatants and precipitates and analyzed for the polyadenylated H3.3 histone mRNA (upper panel) or the replication-dependent H3.2-614 mRNA (middle panel) by S1 nuclease mapping. The same RNA samples were analyzed for the mouse β-actin mRNA using a ribonuclease protection assay (lower panel). Analysis of total RNA or polyribomosal RNA are shown for H3.3 and H3.2 histone mRNAs but not for actin (lanes 1 and 2). A diagram of the S1 nuclease assay is shown below the figure. ( C ). S1 nuclease mapping of total mRNA from the same cells is shown (lane 1). Lanes 7 and 13 shows polyribosomes incubated in buffer and then treated with protein A beads. Lane 14 is marker pUC18 digested with HpaII. A diagram of the S1 nuclease assay is shown below the figure. ( D ). PCR products were detected by ultraviolet (UV) illumination of ethidium bromide stained 2% agarose gels.
    Np 40 Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1 article reviews
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    92
    Promega rnase h
    U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by <t>RNase</t> H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.
    Rnase H, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 90 article reviews
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    Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Journal: Infection and Immunity

    Article Title: Calprotectin Expression by Gingival Epithelial Cells

    doi: 10.1128/IAI.69.5.3248-3254.2001

    Figure Lengend Snippet: Regulation of IL-8 mRNA expression in immortalized human gingival keratinocytes. (A) A representative storage phosphor scan demonstrates that, by using quantitative RNase protection assay, moderate IL-8 mRNA expression was detected in 8 μg of total RNA from immortalized keratinocytes under normal control conditions as seen at time zero. Following treatment of separate flasks of the same culture over 24 h with 50 ng of PMA per ml, IL-8 mRNA expression dramatically increases over a 6-h period, returning to nearly constitutive levels by 12 h. Test lanes, hours 0 to 24, show the protected probes after binding to IL-8 (374 bp) and GAPDH (220 bp) mRNA, respectively, within the sample, followed by RNase treatment. An increase in the band intensity of GAPDH at 2 to 6 h reflects an increase in the total amount of mRNA, as expected with PMA stimulation. This did not affect quantitation (see panel B, below), since the ratio of GAPDH to IL-8 was still proportionally constant. Control lanes (C1 and C2) show the two probes GAPDH (C1, 266 bp) and IL-8 (C2, 420 bp) to which no RNase has been added. The results are representative of three experiments. (B) Quantitation of IL-8 mRNA in immortalized keratinocytes following induction with PMA. From panel A the signal intensity determined for the IL-8 protected fragment was normalized to the abundance of the internal control GAPDH at each time point. The data shown are the mean ± the standard deviation ( n = 3).

    Article Snippet: The radiolabeled probes were synthesized under the following reaction conditions: 500 μM concentrations each of rCTP, rGTP, and rATP, and 1 μM rUTP; 3 μM [α-32 P]UTP (800 Ci/mmol, 10 mCi/ml) (DuPont NEN Research Products, Boston, Mass.); 0.5 μl of PCR template; 1 U of T7 or T3 RNA polymerase (Stratagene); 2 μl of transcription buffer (Stratagene); 40 U of RNase inhibitor (Promega, Madison, Wis.); and distilled H2 O to a total volume of 10 μl.

    Techniques: Expressing, Rnase Protection Assay, Binding Assay, Quantitation Assay, Standard Deviation

    SLBP is bound to histone mRNAs in the cell. ( A ) Polyribosomes isolated from mouse myeloma cells were suspended in buffer containing 10 mM EDTA as described in Materials and Methods. α-SLBP was added in the presence of either 0.1% Tween-20 (lanes 3 and 4) or 0.1% NP-40 (lanes 5 and 6). RNA was prepared from the immunoprecipitates (lanes 3 and 5) and from the supernatant fraction (lanes 4 and 6) and analyzed by S1 nuclease mapping using the histone H3-614 gene as a probe. RNA prepared from polyribosomes was analyzed in lane 2 and yeast tRNA in lane 1. There is a small size differences (2–4 nt) observed between the total RNA (lane 2) and the immunoprecipitated mRNA compared with the RNA that did not immunoprecipitate. The final lane is pUC18 digested with HpaII. ( B ) Immunoprecipitations were performed from mouse myeloma polyribosomes with preimmune serum (pi) (lanes 4 and 9), α-SLBP (lanes 5 and 10), α-SLBP preincubated with 1 μg of antigenic peptide (lanes 6 and 11) or with an unrelated antibody, α-CUL (lanes 7 and 12). RNA was prepared from both supernatants and precipitates and analyzed for the polyadenylated H3.3 histone mRNA (upper panel) or the replication-dependent H3.2-614 mRNA (middle panel) by S1 nuclease mapping. The same RNA samples were analyzed for the mouse β-actin mRNA using a ribonuclease protection assay (lower panel). Analysis of total RNA or polyribomosal RNA are shown for H3.3 and H3.2 histone mRNAs but not for actin (lanes 1 and 2). A diagram of the S1 nuclease assay is shown below the figure. ( C ). S1 nuclease mapping of total mRNA from the same cells is shown (lane 1). Lanes 7 and 13 shows polyribosomes incubated in buffer and then treated with protein A beads. Lane 14 is marker pUC18 digested with HpaII. A diagram of the S1 nuclease assay is shown below the figure. ( D ). PCR products were detected by ultraviolet (UV) illumination of ethidium bromide stained 2% agarose gels.

    Journal: Nucleic Acids Research

    Article Title: SLBP is associated with histone mRNA on polyribosomes as a component of the histone mRNP

    doi: 10.1093/nar/gkh798

    Figure Lengend Snippet: SLBP is bound to histone mRNAs in the cell. ( A ) Polyribosomes isolated from mouse myeloma cells were suspended in buffer containing 10 mM EDTA as described in Materials and Methods. α-SLBP was added in the presence of either 0.1% Tween-20 (lanes 3 and 4) or 0.1% NP-40 (lanes 5 and 6). RNA was prepared from the immunoprecipitates (lanes 3 and 5) and from the supernatant fraction (lanes 4 and 6) and analyzed by S1 nuclease mapping using the histone H3-614 gene as a probe. RNA prepared from polyribosomes was analyzed in lane 2 and yeast tRNA in lane 1. There is a small size differences (2–4 nt) observed between the total RNA (lane 2) and the immunoprecipitated mRNA compared with the RNA that did not immunoprecipitate. The final lane is pUC18 digested with HpaII. ( B ) Immunoprecipitations were performed from mouse myeloma polyribosomes with preimmune serum (pi) (lanes 4 and 9), α-SLBP (lanes 5 and 10), α-SLBP preincubated with 1 μg of antigenic peptide (lanes 6 and 11) or with an unrelated antibody, α-CUL (lanes 7 and 12). RNA was prepared from both supernatants and precipitates and analyzed for the polyadenylated H3.3 histone mRNA (upper panel) or the replication-dependent H3.2-614 mRNA (middle panel) by S1 nuclease mapping. The same RNA samples were analyzed for the mouse β-actin mRNA using a ribonuclease protection assay (lower panel). Analysis of total RNA or polyribomosal RNA are shown for H3.3 and H3.2 histone mRNAs but not for actin (lanes 1 and 2). A diagram of the S1 nuclease assay is shown below the figure. ( C ). S1 nuclease mapping of total mRNA from the same cells is shown (lane 1). Lanes 7 and 13 shows polyribosomes incubated in buffer and then treated with protein A beads. Lane 14 is marker pUC18 digested with HpaII. A diagram of the S1 nuclease assay is shown below the figure. ( D ). PCR products were detected by ultraviolet (UV) illumination of ethidium bromide stained 2% agarose gels.

    Article Snippet: Polyribosomes (5–10 μg of polyribosomal RNA) were diluted into 100 μl of NP-40 buffer [0.1% NP-40, 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM DTT, 10 mM EDTA, 40 U RNAsin (Promega, Madison, WI)].

    Techniques: Isolation, Immunoprecipitation, Nuclease Assay, Incubation, Marker, Polymerase Chain Reaction, Staining

    U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.

    Journal: PLoS Genetics

    Article Title: A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

    doi: 10.1371/journal.pgen.1003856

    Figure Lengend Snippet: U1C depletion results in specific alternative splicing alterations in HeLa cells: Specificity and validation. ( A ) U1C knockdown (kd) in HeLa cells. Whole cell lysates were analyzed by SDS-PAGE and Western blot detecting U1C and γ-tubulin. U1 snRNA steady-state levels were analyzed by Northern blotting with probes specific for U1 snRNA and, as a loading control, U3 snoRNA. HeLa cells after U1C knockdown (ΔC) and luciferase-siRNA treated control cells (ctr) were compared. ( B ) Graphical overview of U1C-dependent alternative splicing targets identified by RNA-Seq analysis. ( C ) U1 snRNA blocking in HeLa cells. The efficiency of U1 snRNA blocking was determined by RNase H protection and silver staining. The positions of the full-length U1 snRNA (U1 uncut), the RNase H-cleaved U1 snRNA (U1 cut), and the U2 snRNA (as a control) are marked on the right. ( D ) Alternative splicing patterns of selected U1C target genes (names above the lanes) were analyzed by RT-PCR, using total RNA from HeLa cells after U1C-knockdown (ctr vs. ΔC) or U1 snRNA blocking (ctr vs. U1). Target-specific primers (arrows in the schematics on the right of the panels) were designed to amplify both alternative splicing isoforms. M , DNA size markers (in bp). Upper panel: Top and lower bands represent exon inclusion and skipping products, respectively; an unspecific product for SNHG5 is marked by open circles between the lanes. Lower panel: For MARCH7 top and lower bands reflect usage of the proximal and distal 5′ splice site, respectively. For UFM1 three alternative 5′ splice sites are activated upon U1C knockdown labeled with 1, 2, and 3 on the right.

    Article Snippet: The efficiency of U1 snRNA inhibition was analyzed by an RNase H protection assay: Whole cell extracts were incubated with 5 µM antisense DNA oligonucleotide ( 5′-CAGGTAAGTAT-3′ ) and 1.5 U RNase H (Promega) for 30 min at 37°C.

    Techniques: SDS Page, Western Blot, Northern Blot, Luciferase, RNA Sequencing Assay, Blocking Assay, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Labeling