recombinant rnase inhibitor  (Thermo Fisher)


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    Name:
    RNase Inhibitor
    Description:
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    Catalog Number:
    n8080119
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher recombinant rnase inhibitor
    RNase Inhibitor ribonuclease inhibitor is a 50 kDa recombinant enzyme used to inhibit RNase activity It does not contain DNase or endonuclease activity Features of this enzyme • Inhibits RNase activity preventing degradation of RNA template• Lacks DNA endonuclease activity for better product yield
    https://www.bioz.com/result/recombinant rnase inhibitor/product/Thermo Fisher
    Average 99 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    recombinant rnase inhibitor - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: The liquid supernatant was collected after centrifugation, precipitated with threefold volume absolute ethyl alcohol for 30 min at 4 °C, centrifuged for 10 min at 12,000 rpm. .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs.

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: Briefly, supernatants were obtained from KMSC after removing debris by centrifugation at 2000g for 15 min. Supernatant was passed through MP selective filters. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Real-time Polymerase Chain Reaction:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: Paragraph title: RNA-Immunoprecipitation (RIP) and qPCR ... Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas).

    Microarray:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: We previously characterized the MVs content of mRNA, by microarray analysis , and of microRNA . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Incubation:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min. .. Supplementary information is available at EMBO reports ).

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada). .. For EMSAs of lysates treated with RNase, 10 µL of 10 mg/mL RNase A (Fermentas Inc; Burlington, Ontario, Canada) was added to 1 mL of lysate and incubated for 1 hr at 4 °C.

    Activity Assay:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. However, some exceptions were reported, for example, the CcPR-4 seemed to have a RNase activity mechanism different from those of the type A, B and C RNases [ ].

    Expressing:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. Treatment with RNase did not affect MVs morphology and surface protein expression, evaluated by FACS analyses, as previously reported .

    Modification:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Western Blot:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas). .. One fourth of the TEV eluate and 25 µg of total protein were subjected to western blotting to verify the affinity purification.

    Electron Microscopy:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Immunoprecipitation:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Immunoprecipitation assays. .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Footprinting:

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A . .. Footprinting analyses were performed with freshly prepared 15 mM lead acetate at room temperature.

    Northern Blot:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Labeling:

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A . .. Footprinting analyses were performed with freshly prepared 15 mM lead acetate at room temperature.

    other:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Transmission Assay:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Sequencing:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: In competition experiments reactions were supplemented with 1 pmol of either YY1 consensus DNA element (sequence above), YY1 mutant consensus DNA element (5′-ACTGGCGCTCCGCGATTATCTTGGCGGCTGGT), unlabelled U(20) RNA, or unlabelled C(20) RNA (5′-CCCCCCCCCCCCCCCCCCCC). .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Affinity Purification:

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation
    Article Snippet: .. Single-step affinity purification was done in the presence of CHX (100 µg ml−1 ) and 80 units/ml of RNase inhibitor (RNasine, Fermentas). ..

    Recombinant:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: The enzymatic tests carried out with the recombinant TcPR-4b revealed that this protein presented both DNase and RNase activities (Figures and ). .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Electrophoretic mobility shift assays with recombinant protein contained 0.5 µM recombinant S. purpuratus YY1 protein, 0.1 pmol labelled probe, 50 mM NaCl, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM DTT, and 5% v/v glycerol in a final volume of 10 µL. .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Immunofluorescence:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Immunofluorescence using Rae1 antibody was carried out as described by ). .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Mutagenesis:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: In competition experiments reactions were supplemented with 1 pmol of either YY1 consensus DNA element (sequence above), YY1 mutant consensus DNA element (5′-ACTGGCGCTCCGCGATTATCTTGGCGGCTGGT), unlabelled U(20) RNA, or unlabelled C(20) RNA (5′-CCCCCCCCCCCCCCCCCCCC). .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Isolation:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Paragraph title: Isolation and characterization of MVs ... In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Electrophoretic Mobility Shift Assay:

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays ... Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Purification:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: Transmission and scanning electron microscopy performed on purified MVs showed their spheroid morphology and confirmed their size . .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: Lead (II)-Footprinting Analysis L7Ae protein was purified as described previously ( ). .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A .

    Article Title: Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus
    Article Snippet: RNA probes were labelled via phosphorylation with γ-32P-ATP and T4 polynucleotide kinase (Fermentas Inc., Burlington, Ontario, Canada), and then purified, according to standard methods . .. Reactions containing RNA probes were additionally supplemented with 1 U of RNase inhibitor (Fermentas Inc., Burlington, Ontario, Canada).

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Microscopy:

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death
    Article Snippet: Samples were analysed both by using the Metasystems (Boston, MA, USA) Zeiss Axioplan 2e with a Zeiss Axiocam HRm digital camera and a Leica (Bannockburn, IL, USA) TCS SP5 confocal microscope. .. For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    FACS:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. Treatment with RNase did not affect MVs morphology and surface protein expression, evaluated by FACS analyses, as previously reported .

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: MP pellets were suspended in PBS, and FACS measured the number/µl. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Plasmid Preparation:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: The mouse PKCζ RNA was obtained by linearization of the pBluescriptKS plasmid (gift of S. Louvet) by SalI or XbaI (Biolabs) and transcription from respectively the T3 or T7 promoter of the vector generating sense 534b SalI or antisense 540b transcripts. .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C.

    Software:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation . .. For MVs size and morphology determination, nanoparticle tracking analysis (NTA) was performed using NanoSight LM10 instrument (NanoSight Ltd., Amesbuty, UK) equipped with the NTA 2.0 analytic software .

    RNA Extraction:

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: The filters were then flushed with an RNA extraction reagent to lyse the captured MPs and release their contents. .. To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.).

    Agarose Gel Electrophoresis:

    Article Title: Microparticles from Kidney-Derived Mesenchymal Stem Cells Act as Carriers of Proangiogenic Signals and Contribute to Recovery from Acute Kidney Injury
    Article Snippet: To abolish the mRNA-dependent effects, MPs were pre-incubated with 1 U/ml RNase (Ambion Inc., Austin, TX, USA) for 1 h at 37°C, as previously described (RNase-treated MPs)., The reaction was stopped by adding 10 U/ml RNase inhibitor (Ambion Inc.). .. The complete degradation of RNA by RNase treatment was confirmed by analyzing total RNA extracted from MPs pre-incubated with RNase on 1% agarose gel (data not shown).

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration.

    In Vitro:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. The antifungal activity of TcPR-4b was verified in vitro on M. perniciosa hyphae (Figure A) and was associated to the increase of mitochondrial O2 - production detected by DHE (Figure C and E).

    Article Title: Alternative Processing as Evolutionary Mechanism for the Origin of Novel Nonprotein Coding RNAs
    Article Snippet: In vitro transcribed RNAs were dephosphorylated by Antarctic Phosphatase treatment (New England BioLabs). .. In brief, 5′-32 P labeled RNAs were heat-denatured at 90 °C for 1 min and immediately chilled on ice for at least 2 min. RNA-L7Ae complex formation was performed in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–KOH (pH 7.0), 200 mM potassium acetate, 1.5 mM magnesium acetate, 2.5 μg/μl tRNA, and 10 U RNase inhibitor (Fermentas); specific concentrations of L7Ae protein are indicated in A .

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. To check the inability of these 3′OH-modified RNAs to be elongated by any radiolabelled ribonucleotide, 100 µg of in vitro Xenopus globin or c-myc transcripts modified at their 3′OH-end (globin 3′H or myc 3′H) or not (globin 3′OH or myc 3′OH) were incubated in a standard tailing reaction (Ambion) containing 1 mM ATP, RNase Inhibitor (30 U), [α–32 P] rATP (0.3 µM, 10 mCi, Perkin Elmer) and E. Coli poly(A) polymerase (12 U) in a final 200 µL reaction during 30 min at 22°C.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Ethanol Precipitation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for CIP (see recipe) RNA substrate from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 40 U/µl RNase inhibitor (Thermo Scientific) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) G50 buffer (see recipe) 10 × buffer for T4 PNK forward reaction (see recipe) 10 µCi/µl [γ32 P]ATP (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) .. Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for CIP 7 µl distilled deionized H2 O 8 µl (10 µg) RNA substrate 1 µl 40 U/µl RNase inhibitor 2 µl 1 U/µl CIP.

    Produced:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: C-myc Xenopus RNA was produced in vitro using T7 RNA polymerase and the template pXLmyc digested with HindIII to generate the sense 2.2 kb full-lenght transcript. .. Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C.

    Concentration Assay:

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Migration:

    Article Title: Evidence for an RNA Polymerization Activity in Axolotl and Xenopus Egg Extracts
    Article Snippet: Briefly, 20 µg of in vitro RNA (Xenopus globin or c-myc) were incubated with RNase Inhibitor (20 U; Fermentas), 5X poly(A) polymerase buffer, 2.5 mM MnCl2 , 20 mM cordycepin 5′-triphosphate and E. Coli poly(A) polymerase (8 U) in a 100 µL final reaction during 1 hr at 37°C. .. The size and quality of the different DNA or RNA templates were respectively controlled on a native agarose gel (DNA) or on a formaldehyde gel (RNA) then by electrophoretic migration.

    Staining:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: The morphological analyses performed on MV suspension after staining with propidium iodide did not show the presence of apoptotic bodies. .. In selected experiments MVs were treated with 5U RNase (Ambion Inc., Austin, TX, USA) for 3 h at 37°C; the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) and MVs were washed by ultracentrifugation .

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  • 99
    Thermo Fisher human rac1
    Design and validation of an HTS-compatible assay measuring the biochemical activity of MgcRacGAP. A) Wild type <t>Rac1</t> cycles between GDP- and GTP-bound forms, but owing to a very high affinity towards the guanine nucleotides, the exchange step is highly rate limiting (red arrow). B) Rac1(F28L) exhibits a reduced affinity for nucleotide, and therefore, a higher rate of nucleotide exchange compared to wild type Rac1. Thus, with Rac1(F28L) the GTPase step is rate limiting (red arrow) producing a sensitive, multiple turnover GAP assay. C) A comparison of fluorescence-mediated detection of GDP released from the GAP assay using wild type Rac1 or Rac1(F28L) shows that the fast-cycling mutant Rac1(F28L) allows for sensitive detection of GDP (GAP activity). Error bars represent SD (n=7 for each condition; p
    Human Rac1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human rac1/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    human rac1 - by Bioz Stars, 2020-04
    99/100 stars
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    99
    Thermo Fisher rnaseout recombinant ribonuclease inhibitor
    Design and validation of an HTS-compatible assay measuring the biochemical activity of MgcRacGAP. A) Wild type <t>Rac1</t> cycles between GDP- and GTP-bound forms, but owing to a very high affinity towards the guanine nucleotides, the exchange step is highly rate limiting (red arrow). B) Rac1(F28L) exhibits a reduced affinity for nucleotide, and therefore, a higher rate of nucleotide exchange compared to wild type Rac1. Thus, with Rac1(F28L) the GTPase step is rate limiting (red arrow) producing a sensitive, multiple turnover GAP assay. C) A comparison of fluorescence-mediated detection of GDP released from the GAP assay using wild type Rac1 or Rac1(F28L) shows that the fast-cycling mutant Rac1(F28L) allows for sensitive detection of GDP (GAP activity). Error bars represent SD (n=7 for each condition; p
    Rnaseout Recombinant Ribonuclease Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnaseout recombinant ribonuclease inhibitor/product/Thermo Fisher
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    rnaseout recombinant ribonuclease inhibitor - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    Design and validation of an HTS-compatible assay measuring the biochemical activity of MgcRacGAP. A) Wild type Rac1 cycles between GDP- and GTP-bound forms, but owing to a very high affinity towards the guanine nucleotides, the exchange step is highly rate limiting (red arrow). B) Rac1(F28L) exhibits a reduced affinity for nucleotide, and therefore, a higher rate of nucleotide exchange compared to wild type Rac1. Thus, with Rac1(F28L) the GTPase step is rate limiting (red arrow) producing a sensitive, multiple turnover GAP assay. C) A comparison of fluorescence-mediated detection of GDP released from the GAP assay using wild type Rac1 or Rac1(F28L) shows that the fast-cycling mutant Rac1(F28L) allows for sensitive detection of GDP (GAP activity). Error bars represent SD (n=7 for each condition; p

    Journal: Combinatorial Chemistry & High Throughput Screening

    Article Title: Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP

    doi: 10.2174/1386207318666141205112730

    Figure Lengend Snippet: Design and validation of an HTS-compatible assay measuring the biochemical activity of MgcRacGAP. A) Wild type Rac1 cycles between GDP- and GTP-bound forms, but owing to a very high affinity towards the guanine nucleotides, the exchange step is highly rate limiting (red arrow). B) Rac1(F28L) exhibits a reduced affinity for nucleotide, and therefore, a higher rate of nucleotide exchange compared to wild type Rac1. Thus, with Rac1(F28L) the GTPase step is rate limiting (red arrow) producing a sensitive, multiple turnover GAP assay. C) A comparison of fluorescence-mediated detection of GDP released from the GAP assay using wild type Rac1 or Rac1(F28L) shows that the fast-cycling mutant Rac1(F28L) allows for sensitive detection of GDP (GAP activity). Error bars represent SD (n=7 for each condition; p

    Article Snippet: The F28L variant of human Rac1 was produced by PCR-based Phusion Site-Directed Mutagenesis (Thermo Scientific) and subcloned into the bacterial expression vector pGEX-4T-1 [ ].

    Techniques: Activity Assay, GAP Assay, Fluorescence, Mutagenesis

    Selective, dose-dependent biochemical inhibition of MgcRacGAP by MINC1. A) Molecular structure of MINC1, CID 744230. B) Molecular structure of MINC2, CID 251705. C) MINC1, identified from single dose testing, was subjected to dose response analyses (0.1-100 µM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. MINC1 exhibited a highly selective inhibition of MgcRacGAP, IC 50 15±5 µM. Error bars represent SD (n=8 for each condition). D) MINC2 exhibited a selective inhibition of MgcRacGAP, IC 50 18±7 µM. Error bars represent SD (n=3 for each condition). E) Dose response curves shift if the proteins are pre-incubated with MINC1 before GTP addition, resulting in a decreased IC 50 by almost 1-order of magnitude. IC 50 , no incubation 15±5 µM; IC50, incubation 2±1 µM. Error bars represent SD (n=8 for each condition). E) MINC2 identified from single dose testing was subjected to dose response analyses (0.1-100 μM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. F) Dose response curves shift due to pre-incubation of Rac1 with MINC1. The presence of MgcRacGAP in the mixture had no additive effect on the inhibition. Error bars represent SD (n=3 for each condition). G) MINC1 stabilizes Rac1-MgcRacGAP complex. Pre-treatment of the complex with MINC1 slowed down the dissociation process in dose dependent matter. Error bars not presented (n=2 for each condition) (color image is available online).

    Journal: Combinatorial Chemistry & High Throughput Screening

    Article Title: Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP

    doi: 10.2174/1386207318666141205112730

    Figure Lengend Snippet: Selective, dose-dependent biochemical inhibition of MgcRacGAP by MINC1. A) Molecular structure of MINC1, CID 744230. B) Molecular structure of MINC2, CID 251705. C) MINC1, identified from single dose testing, was subjected to dose response analyses (0.1-100 µM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. MINC1 exhibited a highly selective inhibition of MgcRacGAP, IC 50 15±5 µM. Error bars represent SD (n=8 for each condition). D) MINC2 exhibited a selective inhibition of MgcRacGAP, IC 50 18±7 µM. Error bars represent SD (n=3 for each condition). E) Dose response curves shift if the proteins are pre-incubated with MINC1 before GTP addition, resulting in a decreased IC 50 by almost 1-order of magnitude. IC 50 , no incubation 15±5 µM; IC50, incubation 2±1 µM. Error bars represent SD (n=8 for each condition). E) MINC2 identified from single dose testing was subjected to dose response analyses (0.1-100 μM) against MgcRacGAP (red), p50RhoGAP (green) and BCR GAP (blue) in the biochemical assay. F) Dose response curves shift due to pre-incubation of Rac1 with MINC1. The presence of MgcRacGAP in the mixture had no additive effect on the inhibition. Error bars represent SD (n=3 for each condition). G) MINC1 stabilizes Rac1-MgcRacGAP complex. Pre-treatment of the complex with MINC1 slowed down the dissociation process in dose dependent matter. Error bars not presented (n=2 for each condition) (color image is available online).

    Article Snippet: The F28L variant of human Rac1 was produced by PCR-based Phusion Site-Directed Mutagenesis (Thermo Scientific) and subcloned into the bacterial expression vector pGEX-4T-1 [ ].

    Techniques: Inhibition, Incubation

    Identification of an MgcRacGAP inhibitor through a novel type of screening assay. A) Z’-factor values from plates used in the primary screen. One plate with Z’-factor value lower than 0.5 was excluded from further analysis (#14, Z’-factor -13.55). All of the remaining plates showed Z’-factor values > 0.68. B) A screen of 20,480 chemically diverse compounds (ChemDiv) at 20 µM. The dotted black line represents the three SDs hit cutoff. MINC1 is shown as green dot and red dots show the remaining 244 primary hit compounds. C) Orthogonal screening results to exclude hits resulting from inhibition of reagents of the primary assay detection system. Malachite green was used to detect GTPase turnover, and 37 compounds, shown as green (MINC1) and red dots, resulted in ≥ 30% inhibition (3.5xSD, represented by the dotted black line). D) Counter screen for selectivity. BCR GAP domain was used to exclude compounds that target either Rac1 or RhoGAPs in general. Of the 37 compounds testing positive through the orthogonal screen (C), 8 compounds, at 10 μM, shown as red dots and MINC1 as green dot, resulted in high inhibition of MgcRacGAP ( > 50%, vertical dotted black line) and low inhibition of BCR GAP (

    Journal: Combinatorial Chemistry & High Throughput Screening

    Article Title: Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP

    doi: 10.2174/1386207318666141205112730

    Figure Lengend Snippet: Identification of an MgcRacGAP inhibitor through a novel type of screening assay. A) Z’-factor values from plates used in the primary screen. One plate with Z’-factor value lower than 0.5 was excluded from further analysis (#14, Z’-factor -13.55). All of the remaining plates showed Z’-factor values > 0.68. B) A screen of 20,480 chemically diverse compounds (ChemDiv) at 20 µM. The dotted black line represents the three SDs hit cutoff. MINC1 is shown as green dot and red dots show the remaining 244 primary hit compounds. C) Orthogonal screening results to exclude hits resulting from inhibition of reagents of the primary assay detection system. Malachite green was used to detect GTPase turnover, and 37 compounds, shown as green (MINC1) and red dots, resulted in ≥ 30% inhibition (3.5xSD, represented by the dotted black line). D) Counter screen for selectivity. BCR GAP domain was used to exclude compounds that target either Rac1 or RhoGAPs in general. Of the 37 compounds testing positive through the orthogonal screen (C), 8 compounds, at 10 μM, shown as red dots and MINC1 as green dot, resulted in high inhibition of MgcRacGAP ( > 50%, vertical dotted black line) and low inhibition of BCR GAP (

    Article Snippet: The F28L variant of human Rac1 was produced by PCR-based Phusion Site-Directed Mutagenesis (Thermo Scientific) and subcloned into the bacterial expression vector pGEX-4T-1 [ ].

    Techniques: Screening Assay, Inhibition