rnase  (Worthington Biochemical)


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  • 92
    Name:
    Ribonuclease A
    Description:
    A chromatographically purified lyophilized powder
    Catalog Number:
    LS003431
    Price:
    75
    Source:
    Bovine Pancreas
    Size:
    200 mg
    Cas Number:
    9001.99.4
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    Structured Review

    Worthington Biochemical rnase
    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with <t>DNase/RNase.</t> SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p
    A chromatographically purified lyophilized powder
    https://www.bioz.com/result/rnase/product/Worthington Biochemical
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase - by Bioz Stars, 2021-10
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    Images

    1) Product Images from "Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts"

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1689-6

    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p
    Figure Legend Snippet: et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Techniques Used: Expressing, Acetylene Reduction Assay

    2) Product Images from "Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts"

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-018-1689-6

    et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p
    Figure Legend Snippet: et-1 , tlr2 and tlr3 expression levels in fibroblasts stimulated with SSc-ICs or NHS-ICs pretreated with DNase/RNase. SSc-ICs treated with DNase I (20 KU/ml) or RNase (8 μg/ml) and then added to fibroblast cultures. a ATA-ICs, ACA-ICs and anti-Th/To-ICs on et-1 ; b ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr2 ; c ATA-ICs and anti-Th/To-ICs on ifn-α ; d ATA-ICs, ACA-ICs, ARA-ICs and anti-Th/To-ICs on tlr3 . * p

    Techniques Used: Expressing, Acetylene Reduction Assay

    Related Articles

    Purification:

    Article Title: Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity
    Article Snippet: .. Purified bovine RNase B and RNase A mixture (234.7 mg; Worthington Biochemical Corp.) was dissolved in tris buffer [100 mM (pH 8.0)], to which 8.8 mg of pronase (EMD Millipore) was added. ..

    Incubation:

    Article Title: Isolation and Characterization of Chemo-Resistant Stem Cells from a Mouse Model of Hereditary Non-Polyposis Colon Cancer
    Article Snippet: .. Briefly, the cell suspension was fixed in ice cold 80% EtOH for 1 h, incubated with RNAse (Worthington Biochemical Corporation, Freehold, NJ, USA) for 20 mins at 37°C and stained with 50 µg/mL propidium iodide (PI, Calbiochem Research Chemicals, La Jolla, CA, USA) in the dark overnight at 4°C. ..

    Article Title: Plasma cell proliferative index is an independent predictor of progression in smoldering multiple myeloma
    Article Snippet: .. This was followed by the wash step and the incubation in the 1000 units/mL RNAse in PBS (Worthington Biochemical Corporation). ..

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts
    Article Snippet: .. DNase and RNase treatment SSc-ICs were incubated for 1 h at 37 °C with recombinant DNase I or RNase (20 KU/ml and 8 μg/ml, respectively; Worthington Biochemical Corporation, Lakewood, NJ, USA) and then added to cells for 24 h. RT-PCR for tlr2 , tlr3 , ifn -α and et-1 was then performed. ..

    Staining:

    Article Title: Isolation and Characterization of Chemo-Resistant Stem Cells from a Mouse Model of Hereditary Non-Polyposis Colon Cancer
    Article Snippet: .. Briefly, the cell suspension was fixed in ice cold 80% EtOH for 1 h, incubated with RNAse (Worthington Biochemical Corporation, Freehold, NJ, USA) for 20 mins at 37°C and stained with 50 µg/mL propidium iodide (PI, Calbiochem Research Chemicals, La Jolla, CA, USA) in the dark overnight at 4°C. ..

    other:

    Article Title: Potential of Soluble Decellularized Extracellular Matrix for Musculoskeletal Tissue Engineering – Comparison of Various Mesenchymal Tissues
    Article Snippet: Followed by three washes in PBS at 4°C for 30 min each, pieces of tissues were transferred to 40 ml of Hanks Buffered Salt Solution (HBSS, Thermo Fisher Scientific, Pittsburgh, PA, United States) supplemented with 200 U/ml DNase and 50 U/ml RNase (Worthington, Lakewood, NJ, United States) with continuous agitation at 37°C for 24 h. Finally, pieces of tissues were washed six times in PBS, as above.

    Centrifugation:

    Article Title: Discovering de novo peptide substrates for enzymes using machine learning
    Article Snippet: .. The cells were harvested by centrifugation at 2000 × g for 30 min, resuspended in lysis buffer (50 mM Tris-HCl buffer, pH 7.5, with 250 mM NaCl), and supplemented with 0.1 mg mL−1 lysozyme (Worthington Biochemical Corp), 5 µg mL−1 DNAse I (Sigma), and 5 µg mL−1 RNAse (Worthington Biochemical Corp.). ..

    Article Title: Discovering de novo peptide substrates for enzymes using machine learning
    Article Snippet: .. The cells were harvested by centrifugation at 2000 × g for 30 min, resuspended in lysis buffer (50 mM Tris-HCl buffer, pH 7.5, with 250 mM NaCl), and supplemented with 0.1 mg mL−1 lysozyme (Worthington Biochemical Corp), 5 µg mL−1 DNAse I (Sigma), and 5 µg mL−1 RNAse (Worthington Biochemical Corp.). ..

    Lysis:

    Article Title: Discovering de novo peptide substrates for enzymes using machine learning
    Article Snippet: .. The cells were harvested by centrifugation at 2000 × g for 30 min, resuspended in lysis buffer (50 mM Tris-HCl buffer, pH 7.5, with 250 mM NaCl), and supplemented with 0.1 mg mL−1 lysozyme (Worthington Biochemical Corp), 5 µg mL−1 DNAse I (Sigma), and 5 µg mL−1 RNAse (Worthington Biochemical Corp.). ..

    Article Title: Discovering de novo peptide substrates for enzymes using machine learning
    Article Snippet: .. The cells were harvested by centrifugation at 2000 × g for 30 min, resuspended in lysis buffer (50 mM Tris-HCl buffer, pH 7.5, with 250 mM NaCl), and supplemented with 0.1 mg mL−1 lysozyme (Worthington Biochemical Corp), 5 µg mL−1 DNAse I (Sigma), and 5 µg mL−1 RNAse (Worthington Biochemical Corp.). ..

    Labeling:

    Article Title: Biochemical Methods to Characterize RNA Polymerase II Elongation Complexes
    Article Snippet: .. Transcription buffer (50 mM HEPES-KOH pH 7.5, 100 mM KCl, 1 mM MnCl2 , 0.5 mM DTT, 10% glycerol, 0.3 mM UpG, 0.1 μg/μl BSA, 1 unit/μl RNasin) 5-Bromo-UTP (Sigma-Aldrich) Nucleotide labeling mix (4 mM ATP, 2 mM 5-Bromo-UTP, 0.05 mM CTP, 5 μl of alpha-32 P-CTP 6000 Ci/mmol, MP Biomedical) 10X DNase I buffer (100 mM MgCl2 , 50 mM CaCl2 , 100 mM Tris-HCl pH 8.0) DNase I 10 unit/μl (Worthington) RNase A 5 mg/ml 10 mg/ml yeast total RNA ..

    Recombinant:

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts
    Article Snippet: .. DNase and RNase treatment SSc-ICs were incubated for 1 h at 37 °C with recombinant DNase I or RNase (20 KU/ml and 8 μg/ml, respectively; Worthington Biochemical Corporation, Lakewood, NJ, USA) and then added to cells for 24 h. RT-PCR for tlr2 , tlr3 , ifn -α and et-1 was then performed. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts
    Article Snippet: .. DNase and RNase treatment SSc-ICs were incubated for 1 h at 37 °C with recombinant DNase I or RNase (20 KU/ml and 8 μg/ml, respectively; Worthington Biochemical Corporation, Lakewood, NJ, USA) and then added to cells for 24 h. RT-PCR for tlr2 , tlr3 , ifn -α and et-1 was then performed. ..

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  • 95
    Worthington Biochemical dnase i
    Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c)  “Run-on” activity assayed later during fractionation (as in  a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Worthington Biochemical rnase t1
    LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the <t>RNase</t> T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .
    Rnase T1, supplied by Worthington Biochemical, 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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    86
    Worthington Biochemical rnase a
    p50 binds both to the 5′ end of osk ) was incubated and UV cross-linked to proteins in the oocyte extract in the absence of competitor RNA (lane 1 ). Competitions were carried out with a 100-fold excess of either repressor fragment (rep, lane 2 ), Eco RI– Bgl II fragment containing the 5′ activator in sense orientation (5′/act-s, lane 3), or the same Eco RI– Bgl II fragment in antisense orientation (5′/act-as, lane 4 ). Immunoprecipitation of proteins UV cross-linked to radiolabeled repressor fragment (lane 5 ) by anti-Bruno antiserum (lane 6 ) or preimmune serum (lane 7 ). Simultaneous binding of p50 and Bruno was tested by treating with <t>RNase</t> A only after the immunoprecipitation. (Lane 8 ) Anti-Bruno antiserum; (lane 9 ) preimmune serum. We noted that in some cases (as shown here) the intensity of the upper band of the p50 doublet was reduced after coprecipitation. The locations of the 5′ and 3′ competitors in the osk transcript are indicated below. The radiolabeled probe is indicated by an asterisk. The reverse experiment, with activator element RNA as a radioactive probe, and cold activator and repressor RNA elements as unlabeled competitors, yielded the same result with respect to p50 (data not shown).
    Rnase A, supplied by Worthington Biochemical, 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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    Image Search Results


    Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c)  “Run-on” activity assayed later during fractionation (as in  a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

    Journal: Nature methods

    Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

    doi: 10.1038/nmeth.1705

    Figure Lengend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

    Article Snippet: Resuspended nuclei were digested (30 min; 33°C) with either (i) DNase I (protease- and RNase-free; Worthington;10 units/107 cells in 100 μl PB plus 0.5 mM CaCl2 ), or (ii) Hae III (Invitrogen; 1000 units/107 cells), or (iii) Hind III (New England Biolabs; 1000 units/107 cells) in PB; reactions were stopped by adding EDTA to 2.5 mM and cooling in iced water.

    Techniques: Purification, Mass Spectrometry, Activity Assay, Fractionation

    LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

    Journal: Nucleic Acids Research

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

    doi: 10.1093/nar/gkp732

    Figure Lengend Snippet: LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

    Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Chromatography, Mass Spectrometry

    CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

    Journal: Nucleic Acids Research

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

    doi: 10.1093/nar/gkp732

    Figure Lengend Snippet: CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

    Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

    Techniques: Mass Spectrometry, Derivative Assay, Sequencing

    Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

    Journal: Nucleic Acids Research

    Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

    doi: 10.1093/nar/gkp732

    Figure Lengend Snippet: Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

    Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

    Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

    Mt FRS activity. ( A ) Aminoacylation of unmodified M. tuberculosis tRNA Phe . The 3′- 32 P-labeled tRNA transcript was charged with L-Phe in the presence of purified Mt FRS. The extent of aminoacylation was analyzed according to Varshney et al. ( 37 ). Prior to polyacrylamide gel electrophoresis, tRNA and Phe-tRNA were digested with RNase T1 to yield 3′- 32 P-labeled CCACCA and CCACCA-Phe, respectively. (+) and (-) indicate the presence and the absence of the enzyme. (B–D) Synthesis of the Phe-AMP intermediate by Mt FRS in the presence of unmodified M. tuberculosis tRNA Phe ( B ), E. coli tRNA Phe ( C ) and yeast tRNA Phe ( D ). Phe-[ 32 P]AMP synthesis was monitored by thin layer chromatography. Phe-AMP and ATP denote 32 P-labeled compounds. F – full reaction; -E, -tRNA and -Phe – control reactions lacking the enzyme, tRNA or amino acid.

    Journal: Nucleic Acids Research

    Article Title: Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation

    doi: 10.1093/nar/gkab272

    Figure Lengend Snippet: Mt FRS activity. ( A ) Aminoacylation of unmodified M. tuberculosis tRNA Phe . The 3′- 32 P-labeled tRNA transcript was charged with L-Phe in the presence of purified Mt FRS. The extent of aminoacylation was analyzed according to Varshney et al. ( 37 ). Prior to polyacrylamide gel electrophoresis, tRNA and Phe-tRNA were digested with RNase T1 to yield 3′- 32 P-labeled CCACCA and CCACCA-Phe, respectively. (+) and (-) indicate the presence and the absence of the enzyme. (B–D) Synthesis of the Phe-AMP intermediate by Mt FRS in the presence of unmodified M. tuberculosis tRNA Phe ( B ), E. coli tRNA Phe ( C ) and yeast tRNA Phe ( D ). Phe-[ 32 P]AMP synthesis was monitored by thin layer chromatography. Phe-AMP and ATP denote 32 P-labeled compounds. F – full reaction; -E, -tRNA and -Phe – control reactions lacking the enzyme, tRNA or amino acid.

    Article Snippet: Prior to electrophoresis, on a 10% acid/7 M urea polyacrylamide gel, 10 pmoles of uncharged and aminoacylated tRNA (Phe-tRNAPhe) were completely digested with 2 U of RNase T1 (Worthington) in 10 mM sodium acetate (pH 4.5).

    Techniques: Activity Assay, Labeling, Purification, Polyacrylamide Gel Electrophoresis, Thin Layer Chromatography

    p50 binds both to the 5′ end of osk ) was incubated and UV cross-linked to proteins in the oocyte extract in the absence of competitor RNA (lane 1 ). Competitions were carried out with a 100-fold excess of either repressor fragment (rep, lane 2 ), Eco RI– Bgl II fragment containing the 5′ activator in sense orientation (5′/act-s, lane 3), or the same Eco RI– Bgl II fragment in antisense orientation (5′/act-as, lane 4 ). Immunoprecipitation of proteins UV cross-linked to radiolabeled repressor fragment (lane 5 ) by anti-Bruno antiserum (lane 6 ) or preimmune serum (lane 7 ). Simultaneous binding of p50 and Bruno was tested by treating with RNase A only after the immunoprecipitation. (Lane 8 ) Anti-Bruno antiserum; (lane 9 ) preimmune serum. We noted that in some cases (as shown here) the intensity of the upper band of the p50 doublet was reduced after coprecipitation. The locations of the 5′ and 3′ competitors in the osk transcript are indicated below. The radiolabeled probe is indicated by an asterisk. The reverse experiment, with activator element RNA as a radioactive probe, and cold activator and repressor RNA elements as unlabeled competitors, yielded the same result with respect to p50 (data not shown).

    Journal: Genes & Development

    Article Title: Localization-dependent translation requires a functional interaction between the 5? and 3? ends of oskar mRNA

    doi:

    Figure Lengend Snippet: p50 binds both to the 5′ end of osk ) was incubated and UV cross-linked to proteins in the oocyte extract in the absence of competitor RNA (lane 1 ). Competitions were carried out with a 100-fold excess of either repressor fragment (rep, lane 2 ), Eco RI– Bgl II fragment containing the 5′ activator in sense orientation (5′/act-s, lane 3), or the same Eco RI– Bgl II fragment in antisense orientation (5′/act-as, lane 4 ). Immunoprecipitation of proteins UV cross-linked to radiolabeled repressor fragment (lane 5 ) by anti-Bruno antiserum (lane 6 ) or preimmune serum (lane 7 ). Simultaneous binding of p50 and Bruno was tested by treating with RNase A only after the immunoprecipitation. (Lane 8 ) Anti-Bruno antiserum; (lane 9 ) preimmune serum. We noted that in some cases (as shown here) the intensity of the upper band of the p50 doublet was reduced after coprecipitation. The locations of the 5′ and 3′ competitors in the osk transcript are indicated below. The radiolabeled probe is indicated by an asterisk. The reverse experiment, with activator element RNA as a radioactive probe, and cold activator and repressor RNA elements as unlabeled competitors, yielded the same result with respect to p50 (data not shown).

    Article Snippet: Fifty micrograms of RNase A and 4 units of RNase T1 (Worthington Biochemical) were used.

    Techniques: Incubation, Activated Clotting Time Assay, Immunoprecipitation, Binding Assay