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Promega rnasin 1
Rnasin 1, 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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Purification:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1
Article Snippet: For RT of an mRNA subset, 5 µ g of purified RNA was mixed initially with 400 ng of anchor primers dT11GG and dT11CA, kept for 10 minutes at 658C, followed by 10 minutes at room temperature. .. Three hundred units of AMV-RT (GibcoBRL) were added and the reaction was incubated for 1 hour at 42°C in RT buffer containing 10 mM dNTP, 10 mM DTT, and 40 U of RNasin 1 (Promega, Madison, WI), and then incubated at 95°C for 5 minutes.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1
Article Snippet: Paragraph title: Differential Display Reverse Transcription Polymerase Chain Reaction (DD RT-PCR) ... Three hundred units of AMV-RT (GibcoBRL) were added and the reaction was incubated for 1 hour at 42°C in RT buffer containing 10 mM dNTP, 10 mM DTT, and 40 U of RNasin 1 (Promega, Madison, WI), and then incubated at 95°C for 5 minutes.

Incubation:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1
Article Snippet: .. Three hundred units of AMV-RT (GibcoBRL) were added and the reaction was incubated for 1 hour at 42°C in RT buffer containing 10 mM dNTP, 10 mM DTT, and 40 U of RNasin 1 (Promega, Madison, WI), and then incubated at 95°C for 5 minutes. .. PCR was performed with 5 µ l of the cDNA reaction in a PTC-100 Thermal cycler (MJ Research, Waltham, MA) using PCR buffer with 2.5 U of AmpliTaq DNA Polymerase 1 (Perkin-Elmer, Foster City, CA), 1.25 mM MgCl2 , 4 µM dNTP, 1 µ l of S 35 dATP (1300 Ci/mmol; Amersham Pharmacia Biotech, Uppsala, Sweden), 500 ng of the respective downstream anchor primer dT11GG or dT12CA, and 400 ng of the upstream DD primer from the set of 26 decameric oligonucleotide primers according to Bauer et al. [ ].

Polyacrylamide Gel Electrophoresis:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1
Article Snippet: Three hundred units of AMV-RT (GibcoBRL) were added and the reaction was incubated for 1 hour at 42°C in RT buffer containing 10 mM dNTP, 10 mM DTT, and 40 U of RNasin 1 (Promega, Madison, WI), and then incubated at 95°C for 5 minutes. .. The PCR products were resolved on a 6% PAGE/7 M urea gel in 16 TBE buffer.

Polymerase Chain Reaction:

Article Title: Met-HGF/SF Signal Transduction Induces Mimp, a Novel Mitochondrial Carrier Homologue, Which Leads to Mitochondrial Depolarization 1
Article Snippet: Three hundred units of AMV-RT (GibcoBRL) were added and the reaction was incubated for 1 hour at 42°C in RT buffer containing 10 mM dNTP, 10 mM DTT, and 40 U of RNasin 1 (Promega, Madison, WI), and then incubated at 95°C for 5 minutes. .. PCR was performed with 5 µ l of the cDNA reaction in a PTC-100 Thermal cycler (MJ Research, Waltham, MA) using PCR buffer with 2.5 U of AmpliTaq DNA Polymerase 1 (Perkin-Elmer, Foster City, CA), 1.25 mM MgCl2 , 4 µM dNTP, 1 µ l of S 35 dATP (1300 Ci/mmol; Amersham Pharmacia Biotech, Uppsala, Sweden), 500 ng of the respective downstream anchor primer dT11GG or dT12CA, and 400 ng of the upstream DD primer from the set of 26 decameric oligonucleotide primers according to Bauer et al. [ ].

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  • 91
    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
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    99
    Promega rnase inhibitor rnasin
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
    Rnase Inhibitor Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    99
    Promega rnasin
    RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM <t>DTT,</t> and 1 μl of <t>RNasin</t> [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.
    Rnasin, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1171 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

    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Sequencing, Incubation, Modification

    T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Primer Extension Assay, Incubation, Molecular Weight, Marker

    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

    RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.

    Journal: Journal of Virology

    Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

    doi: 10.1128/JVI.77.21.11347-11356.2003

    Figure Lengend Snippet: RNA binding activities of wild-type (WT) and mutant helicase proteins. The wild-type or mutant protein was incubated with labeled short ssRNA under standard reaction conditions. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled 26-mer ssRNA oligonucleotides in 20 μl of helicase reaction buffer (30 mM Tris-HCl [pH 7.5], 3 mM MgCl 2 , 10 mM DTT, and 1 μl of RNasin [Promega]). As a control, labeled RNA without VP6 was incubated in one reaction mixture. The binding reaction mixture was incubated at 37°C for 15 min; then the reaction was terminated by the addition of 5× RNA loading dye containing 0.5% Nonidet P-40, and the mixture was subsequently electrophoresed on a 4% polyacrylamide-0.5× Tris-borate-EDTA gel buffer. The bound complex was visualized by autoradiography. (A) Autoradiogram of PAGE showing the positions of bound RNA of the wild-type and three mutant VP6 proteins and free unbound RNA. An RNA probe without VP6 was also resolved as a control (indicated as RNA). (B) The radioactivity of labeled proteins in each reaction mixture was estimated by phosphorimager as described in Materials and Methods, and the efficiencies of the RNA binding activities of the three mutant proteins were compared with that of the wild-type VP6. The RNA probe control is indicated as C. The figure shows the mean values of the results of three different experiments.

    Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex.

    Techniques: RNA Binding Assay, Mutagenesis, Incubation, Labeling, Binding Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Radioactivity

    Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.

    Journal: Journal of Virology

    Article Title: Defining the Structure-Function Relationships of Bluetongue Virus Helicase Protein VP6

    doi: 10.1128/JVI.77.21.11347-11356.2003

    Figure Lengend Snippet: Analysis of VP6-RNA complexes by gel filtration chromatography. Purified VP6 was incubated with S7 transcript in 30 mM Tris-HCl (pH 7.5), 10 mM DTT, 3 mM MgCl 2 , and 2.5 U of RNasin. The reaction mixture was incubated at 37°C for 30 min. Products were loaded on to an equilibrated Hi-Load Superdex-200 gel filtration column and eluted with same buffer at a flow rate of 0.3 ml/min as described in Materials and Methods. The column was calibrated with a set of globular protein standards (Amersham Pharmacia Biotech) consisting of ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (43 kDa), and myoglobulin (17 kDa) prior to the VP6 loading. The arrows point to the positions of their elution. Note that the three peaks of VP6 eluted near the positions of catalase, aldolase, and albumin, which are equivalent to the hexamer, tetramer, and monomer of VP6, respectively.

    Article Snippet: Briefly, a duplex unwinding reaction was performed in a 16-μl volume with 30 mM Tris-HCl (pH 7.5), 3 mM MgCl2 , 10 mM DTT, 5 mM ATP, 1 μl of RNasin (Promega), 1 nM VP6 protein, and 1 nM 32 P-labeled RNA duplex.

    Techniques: Filtration, Chromatography, Purification, Incubation, Flow Cytometry