rnase t1  (Worthington Biochemical)


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    Worthington Biochemical rnase t1
    Reverse phase HPLC chromatograms of ( A ) control <t>RNase</t> T1 digest of tRNA Phe and ( B ) RNase T1 digest of CMC-reacted tRNA Phe . The three chromatographic peaks at 13.4, 16.4 and 30.0 min in the bottom chromatogram have absorbances that are 2–5 times greater than their counterparts in the control chromatogram.
    Rnase T1, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase t1/product/Worthington Biochemical
    Average 94 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    rnase t1 - by Bioz Stars, 2020-11
    94/100 stars

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    1) Product Images from "Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry"

    Article Title: Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry

    Journal: Nucleic Acids Research

    doi:

    Reverse phase HPLC chromatograms of ( A ) control RNase T1 digest of tRNA Phe and ( B ) RNase T1 digest of CMC-reacted tRNA Phe . The three chromatographic peaks at 13.4, 16.4 and 30.0 min in the bottom chromatogram have absorbances that are 2–5 times greater than their counterparts in the control chromatogram.
    Figure Legend Snippet: Reverse phase HPLC chromatograms of ( A ) control RNase T1 digest of tRNA Phe and ( B ) RNase T1 digest of CMC-reacted tRNA Phe . The three chromatographic peaks at 13.4, 16.4 and 30.0 min in the bottom chromatogram have absorbances that are 2–5 times greater than their counterparts in the control chromatogram.

    Techniques Used: High Performance Liquid Chromatography

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    Worthington Biochemical rnase t1
    Components of the S. pombe <t>RNase</t> MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with <t>RNase</t> T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.
    Rnase T1, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 94/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

    Journal: PLoS ONE

    Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    doi: 10.1371/journal.pone.0112488

    Figure Lengend Snippet: Components of the S. pombe RNase MRP complex. (A) RNA component of S. pombe RNase MRP ( mrp1 ). RNA was separated from the purified RNase MRP with acid-phenol treatment and subjected to 8 M urea-7.5% PAGE (SYBR Gold staining). (B) Nucleotide sequence of S. pombe mrp1 RNA and the fragments used for the sequence analysis. Solid or dashed double-headed arrows show the fragments obtained by digestion with RNase T1 or MazF/PemK RNase, respectively. RNase T1 fragments were identified by Ariadne search program, and PemK/MazF fragments were identified by manual inspection of MS/MS spectra (see also Table S2 ). m 3 Gppp, trimethylguanosine cap. (C) Protein components of S. pombe RNase MRP. The RNase-MRP preparation affinity-purified using FEM3-tagged Rmp1 as bait (Rmp1-FEM3) was separated by SDS-PAGE and visualized with Coomassie Brilliant Blue staining. The proteins identified by LC-MS/MS are shown on the right (see also Table S3 and Figure S6 ). The IgG probably resulted from sloughing from the beads during the affinity purification.

    Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

    Techniques: Purification, Polyacrylamide Gel Electrophoresis, Staining, Sequencing, Mass Spectrometry, Affinity Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy

    Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

    Journal: PLoS ONE

    Article Title: RNase MRP Cleaves Pre-tRNASer-Met in the tRNA Maturation Pathway

    doi: 10.1371/journal.pone.0112488

    Figure Lengend Snippet: Isolation of the catalytically active core of RNase MRP. (A) Urea-PAGE profiles of mrp1 RNA fragments produced by RNase A–mediated limited nucleolysis of RNase MRP (SYBR Gold staining). The S. pombe RNase MRP obtained from a 2-l culture of logarithmically growing cells was digested on FLAG M2 agarose beads at 4°C for 1 h with increasing amounts of RNase A. Lane 1, no RNase A; lane 2, 1 µg/ml; lane 3, 5 µg/ml; lane 4, 10 µg/ml. A portion (5%) of each reaction was loaded per lane. (B) A core of RNase MRP produced by RNase A–mediated limited nucleolysis cleaves pre-tRNA Ser-Met . RNase MRP (pulled down with tagged Rmp1 from JJ095 cells using FLAG M2 agarose) and the mock preparation (pulled down from SP6 cells) were incubated with (+) or without (−) RNase A. Each digested preparation (1 pmol each) was incubated with pre-tRNA Ser-Met (8 pmol) at 37°C for 30 min, and each reaction mixture was subjected to urea-PAGE (SYBR-Gold staining). Band 1, mrp1 RNA; 2, pre-tRNA Ser-Met ; 3, pre-tRNA Ser +trailer; 4, tRNA Met . (C) Double-reciprocal plot of the catalytic reaction of RNase A–mediated partial nucleolysis of RNase MRP. The reaction was performed for 30 min with synthetic pre-tRNA Ser-Met as a substrate under the conditions as in Figure 3B . The plot indicates K M of 0.974 µM and Vmax of 12.9 nM/min. (D) RNA fragments produced by RNase T1 digestion of Band 1 (indicated by solid lines) and Band 2 (broken lines) in (A). The fragments identified by LC-MS/MS are mapped on the mrp1 RNA sequence, where the conserved helices and strands of mrp1 [1] , [29] , [31] are shown in shaded boxes. (E) Nuclease-resistant regions mapped in the secondary structure of mrp1 . The map was according to the previous study [2] with modifications made by the assistance of CentroidHomFold ( http://www.ncrna.org/centroidhomfold ). Dashed-line boxes denote the two putative domains [2] . The nuclease-resistant region is shaded gray. The nucleotides with dotted bar are the consensus sequence ANAGNNA known as the mCR-IV motif [2] . (F) SDS-PAGE profiles of the protein components of RNase MRP before (−) and after (+) RNase A–mediated limited nucleolysis (Coomassie Brilliant Blue staining). The proteins assigned by LC-MS/MS are also shown. Note that the catalytic core of RNase MRP produced by partial nucleolysis contains 8 (underlined) of the 11 total subunits.

    Article Snippet: RNases for in-gel digestion, RNase T1 (Worthington), MazF (Takara Bio), and PemK were further purified before use .

    Techniques: Isolation, Polyacrylamide Gel Electrophoresis, Produced, Staining, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, SDS Page

    Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Journal: Nucleic Acids Research

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    doi: 10.1093/nar/gkt1009

    Figure Lengend Snippet: Template-dependent binding of purified wild-type 3 H-Ile-tRNA 1 and mutant 3 H-Ile-tRNA 1 to ribosomes isolated from Bacillus subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU. ( A ) Wild-type tRNA 1 and ( B ) mutant tRNA 1 sample; note that the mutant sample is a mixture of wild-type tRNA 1 and mutant tRNA 1 . ( C ) The mutant 3 H-Ile-tRNA 1 sample was treated with RNase T1 under native conditions to specifically inactivate the wild-type 3 H-Ile-tRNA 1 Ile by cleavage at G34; the mutant tRNA 1 Ile containing U34 is resistant to this treatment; ( D ) an equimolar amount of non-radioactive competitor Met-tRNA 2 was added to RNase T1-treated mutant tRNA. The oligonucleotide concentration in (C) and (D) was 200 μM. Note that in (D) the mutant tRNA 1 sample was treated with RNase T1 first, followed by aminoacylation with 3 H-Ile, resulting in a doubling of 3 H-Ile-tRNA 1 -specific counts present in the ribosome binding experiments.

    Article Snippet: Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

    Techniques: Binding Assay, Purification, Mutagenesis, Isolation, Concentration Assay

    Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Journal: Nucleic Acids Research

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    doi: 10.1093/nar/gkt1009

    Figure Lengend Snippet: Analysis of tRNA 1 Ile purified from Bacillus subtilis wild-type and mutant strain JJS80. ( A ) Characterization of purified tRNA 1 from B. subtilis wild-type (WT; lanes 1–4) and tRNA 1 from JJS80 (lanes 5–8) by RNase A and T1 analysis. The 5′- 32 P-labeled tRNA was partially digested with 0.004 U of RNase A (lanes 2 and 7) and 10 U of RNase T1 (lanes 3 and 6). Lanes 4 and 5 show a partial alkali digest; lanes 1 and 8 show undigested control reactions. 32 P-labeled fragments were separated by denaturing PAGE and visualized by autoradiography. ( B ) Analysis of purified tRNA 1 and tRNA 1 by 15% native PAGE. tRNAs were visualized by staining with ethidium bromide (EtBr) and northern hybridization using a universal tRNA 1 Ile probe. 0.01 A 260 of total tRNA were applied per lane.

    Article Snippet: Mass spectral analysis of purified isoleucine tRNAs For oligonucleotide sequence analysis, 1 μg of tRNA was digested with 50 U of RNase T1 (Worthington Biochemical Corp.) in 20 mM ammonium acetate, pH 5.3, for 2 h at 37°C.

    Techniques: Purification, Mutagenesis, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Clear Native PAGE, Staining, Northern Blot, Hybridization

    RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Journal: Nucleic Acids Research

    Article Title: Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

    doi: 10.1093/nar/gkp782

    Figure Lengend Snippet: RBM15 and OTT3 are required for general mRNA export. Human 293 cells were transfected with siRNAs targeting RBM15, OTT3 or non-targeting siRNA ( control ) and analyzed at day 2 or 4 posttransfection as indicated. ( A ) RT–qPCR detection of RBM15 and OTT3 transcripts at day 2. Expression levels were calculated from real-time PCR values ( C t ) using relative quantitation method and are plotted on the y -axis after normalization to those obtained in the cells transfected with the non-targeting siRNA control (normalized expression). Mean ( n =3) values are presented, and bars show one SEM. ( B ) Cells were transfected with the indicated siRNAs and, next day, with a plasmid expressing HA-OTT3 (1 μg). At day 2 or day 4 after siRNA transfection, cell pellets were boiled in Laemmli sample buffer, proteins separated on 10% SDS–PAGE and analyzed on western blots with antibodies to RBM15, HA, Ran, β-actin or SR proteins as indicated; or by Coomassie staining. ( C ) Cells at day 2 (left panel) or day 4 (right panel) posttransfection were separated into the C, N1 and N2 fractions, mRNA poly(A) tails were 3′-radiolabeled, cut off by RNase T1 digestion, separated by urea–PAGE and detected by phosphoimager. Positions of size markers (nt) are shown. ( D ) U snRNAs from the same fractions as in (D) were separated by urea–PAGE and detected on northern blots, and positions of the individual U snRNAs are indicated to the left. tRNA was detected on the same gels, by ethidium bromide staining prior to blotting (tRNA).

    Article Snippet: Detection of general mRNA, tRNA and U snRNP For general mRNA detection, poly(A)+ RNA was 3′-labeled with [32 P] pCp (NEN) using T4 RNA ligase (Fermentas), followed by complete digestion with RNase T1 (Worthington).

    Techniques: Transfection, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Plasmid Preparation, SDS Page, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, Northern Blot