gtp  (New England Biolabs)


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    Structured Review

    New England Biolabs gtp
    Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gtp/product/New England Biolabs
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    gtp - by Bioz Stars, 2020-02
    92/100 stars

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    Amplification:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: Isolated DNA fragment was PCR amplified with DNA template-specific primers ( Supplementary Table S2 ) and Q5 High-Fidelity DNA Polymerase (New England Biolabs), following the manufacturer's instructions. .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Agarose Gel Electrophoresis:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: This excised fragment was purified by agarose gel electrophoresis and the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Synthesized:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: RNA was synthesized using a T7 mScript Standard mRNA Production System (CELLSCRIPT, Madison, WI), with 20-μl reactions containing 2 μg of purified DNA template. .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Isolation:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: Isolated DNA fragment was PCR amplified with DNA template-specific primers ( Supplementary Table S2 ) and Q5 High-Fidelity DNA Polymerase (New England Biolabs), following the manufacturer's instructions. .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Purification:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: RNA was synthesized using a T7 mScript Standard mRNA Production System (CELLSCRIPT, Madison, WI), with 20-μl reactions containing 2 μg of purified DNA template. .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Incubation:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs). .. Reactions were incubated for 1 hour at 37 °C and treated with DNase following the manufacturer's instructions.

    Polymerase Chain Reaction:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: The final PCR product was purified as described above and quantified by Qubit fluorometer (Life Technologies). .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

    Gel Extraction:

    Article Title: Reprogramming of Pancreatic Exocrine Cells AR42J Into Insulin-producing Cells Using mRNAs for Pdx1, Ngn3, and MafA Transcription Factors
    Article Snippet: This excised fragment was purified by agarose gel electrophoresis and the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). .. We used a custom ribonucleotide blend comprising 3′-0-Me-m7G(5′)ppp(5′)G ARCA cap analog, pseudouridine triphosphate, 5-methylcytidine triphosphate (TriLink Biotechnologies, San Diego, CA), adenosine triphosphate, and guanosine triphosphate (New England Biolabs).

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  • 93
    New England Biolabs α 32 p gtp
    Purification and characterization of FluPol C a , Elution profile of FluPol C , after affinity purification over IgG-sepharose, from a size-exclusion chromatography column. Eluted protein was detected by measuring the absorbance at 280 nm. b , Fractions corresponding to the major peak eluting from the size-exclusion chromatography column were mixed and analysed by SDS–PAGE on a 15% polyacrylamide gel, alongside the indicated molecular mass markers. Protein was visualized by Coomassie blue staining (PB1, 86.0 kDa; PB2, 87.2 kDa; P3, 81.9 kDa). c , Transcription and replication initiation assays. Lanes 2–6 test for transcription initiation. With the addition of vRNA promoter only (lanes 4 and 5), FluPol C can cleave a capped and radiolabelled 20-nucleotide RNA, demonstrating promoter-dependent endonuclease activity. Lane 6 shows that with the addition of NTPs, this capped primer can be extended to produce a capped transcript, thus demonstrating transcription initiation activity. This result is confirmed by lanes 7–9, which test for extension of a capped and radiolabelled 11-nucleotide RNA primer. Extension only takes place when the polymerase is supplied with NTPs and promoter RNA (lane 9). Lanes 10–12 assay for replication initiation. Lane 12 shows that FluPol C (400 ng per reaction) is able to synthesize ApG dinucleotide in a primer-independent manner. This demonstrates de novo . d , De novo initiation and elongation assay. FluPol C (800 ng) was incubated for 3 h with NTPs, [α- 32 <t>P]GTP</t> and 5′ or 3′ vRNA promoter strands, as indicated. In the presence of both promoter strands (lane 4), FluPol C is able to produce a full-length copy of the template (14 nucleotides, corresponding to the major band), demonstrating de novo replication initiation and elongation activity. The minor slower and faster bands may correspond to non-templated extension and premature termination products, respectively.
    α 32 P Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p gtp/product/New England Biolabs
    Average 93 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    α 32 p gtp - by Bioz Stars, 2020-02
    93/100 stars
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    80
    New England Biolabs ranq69l gtp
    Exp5 binding to pre-miR-30 mutants with different stem lengths. Gel-shift assays were performed as described in Materials and Methods. Lane 1, 32 P-labeled WT pre-miR-30 probe in the absence of <t>Exp5/RanQ69L-GTP;</t> lanes 2–17, with Exp5/RanQ69L-GTP; lanes 3–17, 10 or 40 ng of the indicated unlabeled RNAs were used as competitors. Binding efficiencies (%) were calculated as the intensities of the shifted bands, quantified by a PhosphorImager, divided by that of the shifted band in lane 2 (without competitors). The sequences and predicted secondary structures of the WT and mutant pre-miR-30 RNAs are presented below the autoradiograph. Since SP6 RNA polymerase starts transcription with a ‘G’, the corresponding position in pre-miR-30 was changed from ‘U’ to ‘G’, and its base pairing nucleotide was changed from ‘A’ to ‘C’. These changes are indicated by a box. The 2 nt 3′ overhang was also changed to ‘UU’. These changes did not affect Exp5 binding (data not shown).
    Ranq69l Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
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    83
    New England Biolabs m gtp
    Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in <t>RRL</t> with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of <t>m7-GTP</t> in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).
    M Gtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 83/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m gtp/product/New England Biolabs
    Average 83 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Purification and characterization of FluPol C a , Elution profile of FluPol C , after affinity purification over IgG-sepharose, from a size-exclusion chromatography column. Eluted protein was detected by measuring the absorbance at 280 nm. b , Fractions corresponding to the major peak eluting from the size-exclusion chromatography column were mixed and analysed by SDS–PAGE on a 15% polyacrylamide gel, alongside the indicated molecular mass markers. Protein was visualized by Coomassie blue staining (PB1, 86.0 kDa; PB2, 87.2 kDa; P3, 81.9 kDa). c , Transcription and replication initiation assays. Lanes 2–6 test for transcription initiation. With the addition of vRNA promoter only (lanes 4 and 5), FluPol C can cleave a capped and radiolabelled 20-nucleotide RNA, demonstrating promoter-dependent endonuclease activity. Lane 6 shows that with the addition of NTPs, this capped primer can be extended to produce a capped transcript, thus demonstrating transcription initiation activity. This result is confirmed by lanes 7–9, which test for extension of a capped and radiolabelled 11-nucleotide RNA primer. Extension only takes place when the polymerase is supplied with NTPs and promoter RNA (lane 9). Lanes 10–12 assay for replication initiation. Lane 12 shows that FluPol C (400 ng per reaction) is able to synthesize ApG dinucleotide in a primer-independent manner. This demonstrates de novo . d , De novo initiation and elongation assay. FluPol C (800 ng) was incubated for 3 h with NTPs, [α- 32 P]GTP and 5′ or 3′ vRNA promoter strands, as indicated. In the presence of both promoter strands (lane 4), FluPol C is able to produce a full-length copy of the template (14 nucleotides, corresponding to the major band), demonstrating de novo replication initiation and elongation activity. The minor slower and faster bands may correspond to non-templated extension and premature termination products, respectively.

    Journal: Nature

    Article Title: Crystal structure of the RNA-dependent RNA polymerase from influenza C virus

    doi: 10.1038/nature15525

    Figure Lengend Snippet: Purification and characterization of FluPol C a , Elution profile of FluPol C , after affinity purification over IgG-sepharose, from a size-exclusion chromatography column. Eluted protein was detected by measuring the absorbance at 280 nm. b , Fractions corresponding to the major peak eluting from the size-exclusion chromatography column were mixed and analysed by SDS–PAGE on a 15% polyacrylamide gel, alongside the indicated molecular mass markers. Protein was visualized by Coomassie blue staining (PB1, 86.0 kDa; PB2, 87.2 kDa; P3, 81.9 kDa). c , Transcription and replication initiation assays. Lanes 2–6 test for transcription initiation. With the addition of vRNA promoter only (lanes 4 and 5), FluPol C can cleave a capped and radiolabelled 20-nucleotide RNA, demonstrating promoter-dependent endonuclease activity. Lane 6 shows that with the addition of NTPs, this capped primer can be extended to produce a capped transcript, thus demonstrating transcription initiation activity. This result is confirmed by lanes 7–9, which test for extension of a capped and radiolabelled 11-nucleotide RNA primer. Extension only takes place when the polymerase is supplied with NTPs and promoter RNA (lane 9). Lanes 10–12 assay for replication initiation. Lane 12 shows that FluPol C (400 ng per reaction) is able to synthesize ApG dinucleotide in a primer-independent manner. This demonstrates de novo . d , De novo initiation and elongation assay. FluPol C (800 ng) was incubated for 3 h with NTPs, [α- 32 P]GTP and 5′ or 3′ vRNA promoter strands, as indicated. In the presence of both promoter strands (lane 4), FluPol C is able to produce a full-length copy of the template (14 nucleotides, corresponding to the major band), demonstrating de novo replication initiation and elongation activity. The minor slower and faster bands may correspond to non-templated extension and premature termination products, respectively.

    Article Snippet: Capped and radiolabelled RNA was produced by incubating 5′ diphosphate synthetic 20-nucleotide (5′-ppAAUCUAUAAUAGCAUUAUCC-3′) , or 11-nucleotide (5′-ppGAAUACUCAAG-3′) , RNA (Chemgenes), with [α-32 P]GTP, vaccinia virus capping enzyme (NEB) and 2′- O -methyltransferase (NEB), following the manufacturer’s instructions.

    Techniques: Purification, Affinity Purification, Size-exclusion Chromatography, SDS Page, Staining, Activity Assay, Incubation

    Exp5 binding to pre-miR-30 mutants with different stem lengths. Gel-shift assays were performed as described in Materials and Methods. Lane 1, 32 P-labeled WT pre-miR-30 probe in the absence of Exp5/RanQ69L-GTP; lanes 2–17, with Exp5/RanQ69L-GTP; lanes 3–17, 10 or 40 ng of the indicated unlabeled RNAs were used as competitors. Binding efficiencies (%) were calculated as the intensities of the shifted bands, quantified by a PhosphorImager, divided by that of the shifted band in lane 2 (without competitors). The sequences and predicted secondary structures of the WT and mutant pre-miR-30 RNAs are presented below the autoradiograph. Since SP6 RNA polymerase starts transcription with a ‘G’, the corresponding position in pre-miR-30 was changed from ‘U’ to ‘G’, and its base pairing nucleotide was changed from ‘A’ to ‘C’. These changes are indicated by a box. The 2 nt 3′ overhang was also changed to ‘UU’. These changes did not affect Exp5 binding (data not shown).

    Journal: Nucleic Acids Research

    Article Title: Structural requirements for pre-microRNA binding and nuclear export by Exportin 5

    doi: 10.1093/nar/gkh824

    Figure Lengend Snippet: Exp5 binding to pre-miR-30 mutants with different stem lengths. Gel-shift assays were performed as described in Materials and Methods. Lane 1, 32 P-labeled WT pre-miR-30 probe in the absence of Exp5/RanQ69L-GTP; lanes 2–17, with Exp5/RanQ69L-GTP; lanes 3–17, 10 or 40 ng of the indicated unlabeled RNAs were used as competitors. Binding efficiencies (%) were calculated as the intensities of the shifted bands, quantified by a PhosphorImager, divided by that of the shifted band in lane 2 (without competitors). The sequences and predicted secondary structures of the WT and mutant pre-miR-30 RNAs are presented below the autoradiograph. Since SP6 RNA polymerase starts transcription with a ‘G’, the corresponding position in pre-miR-30 was changed from ‘U’ to ‘G’, and its base pairing nucleotide was changed from ‘A’ to ‘C’. These changes are indicated by a box. The 2 nt 3′ overhang was also changed to ‘UU’. These changes did not affect Exp5 binding (data not shown).

    Article Snippet: Approximately 20 000 c.p.m. of 32 P-labeled RNA was incubated with 2.5 μg Exp5 and 5 μg of RanQ69L-GTP in 15 μl of 1× buffer (NEB buffer 4) at room temperature for 10 min. As a negative control for Exp5/RanQ69L, an equivalent quantity of BSA or RanQ69L-GTP alone was incubated with the RNA.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Autoradiography

    Exp5 binding to pre-miR-30 mutants with different terminal loops. Lane 1, WT pre-miR-30 probe in the absence of Exp5/RanQ69L-GTP; lanes 2–13, with Exp5/RanQ69L-GTP; lanes 3–13, 10 or 40 ng of the indicated unlabeled RNA competitors were added. Binding efficiencies (%) were calculated as the intensities of the shifted bands divided by that of the shifted band in lane 2. Sequences and predicted secondary structures of the loop deletion mutants are shown below the autoradiograph.

    Journal: Nucleic Acids Research

    Article Title: Structural requirements for pre-microRNA binding and nuclear export by Exportin 5

    doi: 10.1093/nar/gkh824

    Figure Lengend Snippet: Exp5 binding to pre-miR-30 mutants with different terminal loops. Lane 1, WT pre-miR-30 probe in the absence of Exp5/RanQ69L-GTP; lanes 2–13, with Exp5/RanQ69L-GTP; lanes 3–13, 10 or 40 ng of the indicated unlabeled RNA competitors were added. Binding efficiencies (%) were calculated as the intensities of the shifted bands divided by that of the shifted band in lane 2. Sequences and predicted secondary structures of the loop deletion mutants are shown below the autoradiograph.

    Article Snippet: Approximately 20 000 c.p.m. of 32 P-labeled RNA was incubated with 2.5 μg Exp5 and 5 μg of RanQ69L-GTP in 15 μl of 1× buffer (NEB buffer 4) at room temperature for 10 min. As a negative control for Exp5/RanQ69L, an equivalent quantity of BSA or RanQ69L-GTP alone was incubated with the RNA.

    Techniques: Binding Assay, Autoradiography

    Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Journal: Nature

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    doi: 10.1038/nature17978

    Figure Lengend Snippet: Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Article Snippet: Reaction was pre-incubated with RRL in the presence of 2 mM GMP-PNP or m GTP and 3 µM RocA or 1% DMSO at 30 °C for 5 min, and then incubated with 50 nM mRNAs at 30 °C for 5 min, followed by reverse transcription with 10 U/μl ProtoScript II (NEB) with 250 nM 5′ 6-FAM labeled primer (5′-6-FAM-ATGCAGAAAAATCACGGC-3′) at 30 °C for 15 min.

    Techniques: Toeprinting Assay, In Vitro, Real-time Polymerase Chain Reaction, Sequencing, Recombinant, Footprinting