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  • 96
    New England Biolabs rtcb ligase
    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, <t>RtcB.</t> We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with <t>T4</t> PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
    Rtcb Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Santa Cruz Biotechnology anti hspc117 faap rtcb
    Reduced AID Binding to IgH S-Regions in DDX1-Depleted Cells CH12 cells transduced with shCtrl or shDDX1 were cultured in CIT-stimulated conditions and analyzed after 24 hr. (A and B) AID ChIP analysis in μ (A) and α (B) regions. Values shown for antibody (IP) or IgG control are expressed as percentage of Input (n = 3, mean ± SD). AID KO CH12 cells were used to determine background levels (dashed line represents probe 2 signal). (C) Western blot of whole-cell and nuclear protein extracts for DDX1 and AID. Purity of nuclear extracts is shown by absence of cytoplasmic Tubulin, and CTCF levels were used as loading control. Amount of nuclear extract loaded is 15 times increased compared to the equivalent amount of whole cell extracts (WCEs). Data are representative of 3 independent experiments. (D and E) Co-immunoprecipitation assays with anti-DDX1 antibody and nuclear protein extracts from (D) AID FLAG-HA or (E) WT CH12 cells CIT stimulated for 24 hr. Western blots were analyzed for DDX1, AID (or FLAG tag), and the tRNA ligase subunit <t>RTCB</t> as a positive control. Representative results from 2 independent pull-downs on each cell type. A. (G) Number of Sμ mutations at each nucleotide, expressed per 10 3 bp (n = 4). Unique Sμ-Sα sequences were amplified from genomic DNA extracted from shCtrl (50 sequences) or shDDX1 (62 sequences) CH12 cells cultured in CIT conditions for 72 hr. .
    Anti Hspc117 Faap Rtcb, supplied by Santa Cruz Biotechnology, 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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    86
    Proteintech rabbit rtcb specific polyclonal
    Reduced AID Binding to IgH S-Regions in DDX1-Depleted Cells CH12 cells transduced with shCtrl or shDDX1 were cultured in CIT-stimulated conditions and analyzed after 24 hr. (A and B) AID ChIP analysis in μ (A) and α (B) regions. Values shown for antibody (IP) or IgG control are expressed as percentage of Input (n = 3, mean ± SD). AID KO CH12 cells were used to determine background levels (dashed line represents probe 2 signal). (C) Western blot of whole-cell and nuclear protein extracts for DDX1 and AID. Purity of nuclear extracts is shown by absence of cytoplasmic Tubulin, and CTCF levels were used as loading control. Amount of nuclear extract loaded is 15 times increased compared to the equivalent amount of whole cell extracts (WCEs). Data are representative of 3 independent experiments. (D and E) Co-immunoprecipitation assays with anti-DDX1 antibody and nuclear protein extracts from (D) AID FLAG-HA or (E) WT CH12 cells CIT stimulated for 24 hr. Western blots were analyzed for DDX1, AID (or FLAG tag), and the tRNA ligase subunit <t>RTCB</t> as a positive control. Representative results from 2 independent pull-downs on each cell type. A. (G) Number of Sμ mutations at each nucleotide, expressed per 10 3 bp (n = 4). Unique Sμ-Sα sequences were amplified from genomic DNA extracted from shCtrl (50 sequences) or shDDX1 (62 sequences) CH12 cells cultured in CIT conditions for 72 hr. .
    Rabbit Rtcb Specific Polyclonal, supplied by Proteintech, 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


    Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Journal: Nature biotechnology

    Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

    doi: 10.1038/s41587-019-0090-6

    Figure Lengend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

    Article Snippet: 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C.

    Techniques: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

    Reduced AID Binding to IgH S-Regions in DDX1-Depleted Cells CH12 cells transduced with shCtrl or shDDX1 were cultured in CIT-stimulated conditions and analyzed after 24 hr. (A and B) AID ChIP analysis in μ (A) and α (B) regions. Values shown for antibody (IP) or IgG control are expressed as percentage of Input (n = 3, mean ± SD). AID KO CH12 cells were used to determine background levels (dashed line represents probe 2 signal). (C) Western blot of whole-cell and nuclear protein extracts for DDX1 and AID. Purity of nuclear extracts is shown by absence of cytoplasmic Tubulin, and CTCF levels were used as loading control. Amount of nuclear extract loaded is 15 times increased compared to the equivalent amount of whole cell extracts (WCEs). Data are representative of 3 independent experiments. (D and E) Co-immunoprecipitation assays with anti-DDX1 antibody and nuclear protein extracts from (D) AID FLAG-HA or (E) WT CH12 cells CIT stimulated for 24 hr. Western blots were analyzed for DDX1, AID (or FLAG tag), and the tRNA ligase subunit RTCB as a positive control. Representative results from 2 independent pull-downs on each cell type. A. (G) Number of Sμ mutations at each nucleotide, expressed per 10 3 bp (n = 4). Unique Sμ-Sα sequences were amplified from genomic DNA extracted from shCtrl (50 sequences) or shDDX1 (62 sequences) CH12 cells cultured in CIT conditions for 72 hr. .

    Journal: Molecular Cell

    Article Title: RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination

    doi: 10.1016/j.molcel.2018.04.001

    Figure Lengend Snippet: Reduced AID Binding to IgH S-Regions in DDX1-Depleted Cells CH12 cells transduced with shCtrl or shDDX1 were cultured in CIT-stimulated conditions and analyzed after 24 hr. (A and B) AID ChIP analysis in μ (A) and α (B) regions. Values shown for antibody (IP) or IgG control are expressed as percentage of Input (n = 3, mean ± SD). AID KO CH12 cells were used to determine background levels (dashed line represents probe 2 signal). (C) Western blot of whole-cell and nuclear protein extracts for DDX1 and AID. Purity of nuclear extracts is shown by absence of cytoplasmic Tubulin, and CTCF levels were used as loading control. Amount of nuclear extract loaded is 15 times increased compared to the equivalent amount of whole cell extracts (WCEs). Data are representative of 3 independent experiments. (D and E) Co-immunoprecipitation assays with anti-DDX1 antibody and nuclear protein extracts from (D) AID FLAG-HA or (E) WT CH12 cells CIT stimulated for 24 hr. Western blots were analyzed for DDX1, AID (or FLAG tag), and the tRNA ligase subunit RTCB as a positive control. Representative results from 2 independent pull-downs on each cell type. A. (G) Number of Sμ mutations at each nucleotide, expressed per 10 3 bp (n = 4). Unique Sμ-Sα sequences were amplified from genomic DNA extracted from shCtrl (50 sequences) or shDDX1 (62 sequences) CH12 cells cultured in CIT conditions for 72 hr. .

    Article Snippet: For immunoblot analysis of RNA pull-down or co-Immunoprecipitation assays the following antibodies were additionally used: anti-FLAG (ThermoScientific), anti-FLAG-HRP (Sigma) and anti- HSPC117/FAAP (RTCB) (Santa Cruz).

    Techniques: Binding Assay, Transduction, Cell Culture, Chromatin Immunoprecipitation, Western Blot, Immunoprecipitation, FLAG-tag, Positive Control, Amplification