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Promega rnasin 40 u
Rnasin 40 U, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 1 article reviews
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rnasin 40 u - by Bioz Stars, 2020-04
93/100 stars

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Spectrophotometry:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: RNA concentration was measured in a Nanodrop UV-Vis Spectrophotometer (Thermo Fisher Scientific). .. After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume).

Purification:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: mRNA quantification by RT-qPCR RNA was extracted from cells growing in exponential phase (24 h post inoculum) with Qiazol and purified according to the manufacturer’s instructions (miRNeasy kit, Qiagen), including an in-column DNase digestion. .. After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume).

Real-time Polymerase Chain Reaction:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume). .. The mix was incubated at RT for 10 min and at 42 °C for 50 min, and then heat-inactivated (70 °C, 10 min). qPCR was done with the 2× GoTaq qPCR Master mix (Promega), 0.15 µM oligos, and 0.5 ng cDNA in a Light Cycler-480 (Roche), in standard conditions, a 384-well format and in at least triplicates.

Concentration Assay:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: RNA concentration was measured in a Nanodrop UV-Vis Spectrophotometer (Thermo Fisher Scientific). .. After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume).

Incubation:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume). .. The mix was incubated at RT for 10 min and at 42 °C for 50 min, and then heat-inactivated (70 °C, 10 min). qPCR was done with the 2× GoTaq qPCR Master mix (Promega), 0.15 µM oligos, and 0.5 ng cDNA in a Light Cycler-480 (Roche), in standard conditions, a 384-well format and in at least triplicates.

Quantitative RT-PCR:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: Paragraph title: mRNA quantification by RT-qPCR ... After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume).

Hybridization:

Article Title: A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants
Article Snippet: .. After hybridization was performed, the RT reaction was set up by adding 4 µl of 5× buffer, 2 µl of dithiothreitol (DTT) at 0.1 M, 1 µl of dNTPs at 10 mM, 1 µl of SCII (Invitrogen), and 1 µl RNasin 40 u µl−1 (Promega) directly to the primed RNA (20 µl final reaction volume). .. The mix was incubated at RT for 10 min and at 42 °C for 50 min, and then heat-inactivated (70 °C, 10 min). qPCR was done with the 2× GoTaq qPCR Master mix (Promega), 0.15 µM oligos, and 0.5 ng cDNA in a Light Cycler-480 (Roche), in standard conditions, a 384-well format and in at least triplicates.

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    Promega rnase h buffer
    <t>RNase</t> H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.
    Rnase H Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h buffer/product/Promega
    Average 91 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
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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 58 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 58 article reviews
    Price from $9.99 to $1999.99
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    86
    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    Image Search Results


    RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H activation by LNA/DNA and 2′- O -methyl gapmers. (Top) Results of RNase H assay. Full-length VR1 mRNA was incubated with a 5-fold excess of oligonucleotides in the presence of RNase H for 7.5 min at 37°C. Substrate (S) and cleavage products (P1 + P2) were separated on a 1.5% agarose gel and stained with ethidium bromide. Lane 1, control; lane 2, DNA 1; lane 3, LNA 9; lane 4, LNA 12; lane 5, LNA 13; lane 6, LNA 14; lane 7, LNA 15; lane 8, OMe 1; Lane 9, OMe 2; lane 10, OMe 3. (Bottom) Quantitative evaluation of the gel. All values are averages and standard deviations of at least three independent experiments and were normalized.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Activation Assay, Rnase H Assay, Incubation, Agarose Gel Electrophoresis, Staining

    RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Journal: Nucleic Acids Research

    Article Title: Design of antisense oligonucleotides stabilized by locked nucleic acids

    doi:

    Figure Lengend Snippet: RNase H assays with a short RNA target. Lane 1, one base ladder produced by alkaline hydrolysis; lane 2, cleavage products after incubation with RNase T1; lane 3, 18mer RNA; lanes 4 and 5, products after incubation of target RNA with all DNA antisense oligonucleotide (DNA 1) in the presence of RNase H after 10 and 30 min, respectively; lanes 6 and 7, products after incubation of target RNA with end-block LNA/DNA/LNA oligonucleotide (LNA 17) in the presence of RNase H after 10 and 30 min; lanes 8 and 9, products after incubation of target RNA with LNA/DNA mixmer (LNA 9) in the presence of RNase H after 10 and 30 min; lanes 10 and 11, products after incubation of target RNA with phosphorothioate in the presence of RNase H after 10 and 30 min. For further details see text.

    Article Snippet: The standard RNase H assay was performed as described previously ( ): 100 nM VR1 mRNA were incubated with a 5-fold excess of an antisense oligonucleotide in a total volume of 10 µl in RNase H buffer (40 mM Tris–HCl pH 7.2, 4 mM MgCl2 , 1 mM DTT, 150 mM NaCl and 1.25 U/µl RNasin; Promega, Madison, WI) for 7.5 min at 37°C in the presence of 0.4 U E.coli RNase H (Promega).

    Techniques: Produced, Incubation, Blocking Assay

    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

    Mutation of amino acid residue H300K impairs both the RNase activity of E rns and the inhibitory effect of E rns on dsRNA-induced MxA expression. (A) Stability of wild-type and mutant E rns as determined by SDS-PAGE. Prestained molecular mass standards (lane 1), 1 μg of wild-type E rns (lane 2), 1 μg of H300K mutant (lane 3), 1 μg of wild-type E rns in maintenance medium (lanes 4 and 5), and 1 μg of H300K mutant in maintenance medium (lanes 6 and 7) are shown. The samples in lanes 5 and 7 were incubated at 37°C for 18 h before electrophoresis. The gel was stained with Coomassie brilliant blue R250. (B) Stability of RNase activity of E rns . Purified wild-type and H300K mutant E rns (bars 1 and 4) and wild-type and mutant E rns incubated in cell culture medium (DMEM plus 2% FCS) for either 10 min at room temperature (bars 2 and 5) or for 18 h at 37°C (bars 3 and 6) were assayed for RNase activity at pH 4.5. The final assay mixture (25 μl) contained wild-type E rns and mutant H300K glycoproteins (0.2 μg), yeast 16-23S RNA (12.5 μg), and RNase inhibitor (RNasin; 40 U) as described in Materials and Methods. The release of acid-soluble RNA was determined by measuring the absorbance at 260 nm. (C) Comparison of dsRNA-induced MxA inhibition by wild-type E rns and H300K mutant. Confluent monolayers of cells were rinsed with maintenance medium and replaced with medium containing 5 μg of poly(rI)-poly(rC)/ml and 1 μg of wild-type E rns /ml (lane 1), 5 μg of poly(rI)-poly(rC)/ml and 1 μg of H300K mutant/ml (lane 2), and 5 μg of poly(rI)-poly(rC)/ml (lane 4). Lane 3, mock treated. Cells were incubated at 37°C for 18 h and subjected to immunoblot analysis for the MxA protein.

    Journal: Journal of Virology

    Article Title: Role for Bovine Viral Diarrhea Virus Erns Glycoprotein in the Control of Activation of Beta Interferon by Double-Stranded RNA

    doi: 10.1128/JVI.78.1.136-145.2004

    Figure Lengend Snippet: Mutation of amino acid residue H300K impairs both the RNase activity of E rns and the inhibitory effect of E rns on dsRNA-induced MxA expression. (A) Stability of wild-type and mutant E rns as determined by SDS-PAGE. Prestained molecular mass standards (lane 1), 1 μg of wild-type E rns (lane 2), 1 μg of H300K mutant (lane 3), 1 μg of wild-type E rns in maintenance medium (lanes 4 and 5), and 1 μg of H300K mutant in maintenance medium (lanes 6 and 7) are shown. The samples in lanes 5 and 7 were incubated at 37°C for 18 h before electrophoresis. The gel was stained with Coomassie brilliant blue R250. (B) Stability of RNase activity of E rns . Purified wild-type and H300K mutant E rns (bars 1 and 4) and wild-type and mutant E rns incubated in cell culture medium (DMEM plus 2% FCS) for either 10 min at room temperature (bars 2 and 5) or for 18 h at 37°C (bars 3 and 6) were assayed for RNase activity at pH 4.5. The final assay mixture (25 μl) contained wild-type E rns and mutant H300K glycoproteins (0.2 μg), yeast 16-23S RNA (12.5 μg), and RNase inhibitor (RNasin; 40 U) as described in Materials and Methods. The release of acid-soluble RNA was determined by measuring the absorbance at 260 nm. (C) Comparison of dsRNA-induced MxA inhibition by wild-type E rns and H300K mutant. Confluent monolayers of cells were rinsed with maintenance medium and replaced with medium containing 5 μg of poly(rI)-poly(rC)/ml and 1 μg of wild-type E rns /ml (lane 1), 5 μg of poly(rI)-poly(rC)/ml and 1 μg of H300K mutant/ml (lane 2), and 5 μg of poly(rI)-poly(rC)/ml (lane 4). Lane 3, mock treated. Cells were incubated at 37°C for 18 h and subjected to immunoblot analysis for the MxA protein.

    Article Snippet: Briefly, the assay mixture (25 μl in total) contained 0.2 μg of wild-type or mutant Erns , 12.5 μg of yeast 16-23S RNA (Worthington), and 40 U of RNase inhibitor (RNasin; Promega) in 20 mM sodium acetate buffer (pH 4.5).

    Techniques: Mutagenesis, Activity Assay, Expressing, SDS Page, Incubation, Electrophoresis, Staining, Purification, Cell Culture, Inhibition

    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