t4 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs t4 pnk
    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or <t>T4</t> PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk/product/New England Biolabs
    Average 95 stars, based on 3163 article reviews
    Price from $9.99 to $1999.99
    t4 pnk - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    90
    Thermo Fisher t4 dna ligase
    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and <t>T4</t> DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 19342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 90 stars, based on 19342 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    79
    Thermo Fisher t 4 kinase
    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and <t>T4</t> DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    T 4 Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t 4 kinase/product/Thermo Fisher
    Average 79 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    t 4 kinase - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    78
    Frontier Scientific h2 t 4 pyp
    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and <t>T4</t> DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    H2 T 4 Pyp, supplied by Frontier Scientific, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h2 t 4 pyp/product/Frontier Scientific
    Average 78 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    h2 t 4 pyp - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    95
    New England Biolabs t4 pdg
    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by <t>T4-PDG.</t> Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively
    T4 Pdg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pdg/product/New England Biolabs
    Average 95 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    t4 pdg - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    99
    Becton Dickinson rpa t4
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    Rpa T4, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 319 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rpa t4/product/Becton Dickinson
    Average 99 stars, based on 319 article reviews
    Price from $9.99 to $1999.99
    rpa t4 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    93
    Enzymatics t4 polymerase
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    T4 Polymerase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polymerase/product/Enzymatics
    Average 93 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    t4 polymerase - by Bioz Stars, 2020-02
    93/100 stars
      Buy from Supplier

    91
    Nexcelom Bioscience t4 cellometer
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    T4 Cellometer, supplied by Nexcelom Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 cellometer/product/Nexcelom Bioscience
    Average 91 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    t4 cellometer - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    99
    Promega t4 polymerase
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    T4 Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polymerase/product/Promega
    Average 99 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    t4 polymerase - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    93
    Roche t4 polymerase
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    T4 Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 polymerase/product/Roche
    Average 93 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    t4 polymerase - by Bioz Stars, 2020-02
    93/100 stars
      Buy from Supplier

    78
    TaKaRa t4 pnk1
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    T4 Pnk1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk1/product/TaKaRa
    Average 78 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    t4 pnk1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    76
    Thermo Fisher pra t4
    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), <t>RPA-T4</t> ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.
    Pra T4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pra t4/product/Thermo Fisher
    Average 76 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pra t4 - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    94
    Thermo Fisher rpa t4
    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs <t>RPA-T4,</t> SK3 and #21, or mouse IgG as
    Rpa T4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rpa t4/product/Thermo Fisher
    Average 94 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    rpa t4 - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    95
    New England Biolabs t4 dna polymerase
    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of <t>T4</t> DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 6750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 95 stars, based on 6750 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    90
    TaKaRa t4 dna polymerase
    The PCR product of a foreign gene was amplified by <t>T4</t> DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    T4 Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/TaKaRa
    Average 90 stars, based on 730 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    90
    Thermo Fisher t4 dna polymerase exonuclease
    UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the <t>T4</t> DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .
    T4 Dna Polymerase Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase exonuclease/product/Thermo Fisher
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase exonuclease - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    90
    Thermo Fisher materials t4 dna ligase
    DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) <t>T4</t> DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.
    Materials T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/materials t4 dna ligase/product/Thermo Fisher
    Average 90 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    materials t4 dna ligase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    90
    TaKaRa t4 dna ligase
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 5426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/TaKaRa
    Average 90 stars, based on 5426 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    77
    Data Sciences International dataquest a r t 4 0
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    Dataquest A R T 4 0, supplied by Data Sciences International, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dataquest a r t 4 0/product/Data Sciences International
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dataquest a r t 4 0 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    99
    BioLegend rpa t4
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    Rpa T4, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rpa t4/product/BioLegend
    Average 99 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    rpa t4 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    PEQLAB cellometer t4
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    Cellometer T4, supplied by PEQLAB, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cellometer t4/product/PEQLAB
    Average 99 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    cellometer t4 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    PerkinElmer 125i t4
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    125i T4, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/125i t4/product/PerkinElmer
    Average 99 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    125i t4 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    94
    Promega ligase t4
    Schematic drawing of major ligation factors and evaluation of <t>T4</t> ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.
    Ligase T4, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ligase t4/product/Promega
    Average 94 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    ligase t4 - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    86
    Millipore hmt3522 t4 2
    Schematic drawing of major ligation factors and evaluation of <t>T4</t> ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.
    Hmt3522 T4 2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hmt3522 t4 2/product/Millipore
    Average 86 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    hmt3522 t4 2 - by Bioz Stars, 2020-02
    86/100 stars
      Buy from Supplier

    95
    Millipore t4 pnk
    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with <t>T4</t> PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
    T4 Pnk, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk/product/Millipore
    Average 95 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    t4 pnk - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    90
    TaKaRa t4 rna ligase
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    T4 Rna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase/product/TaKaRa
    Average 90 stars, based on 733 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    94
    BioLegend rra t4
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    Rra T4, supplied by BioLegend, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rra t4/product/BioLegend
    Average 94 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    rra t4 - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    82
    NEN Life Science i t4
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    I T4, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 82/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/i t4/product/NEN Life Science
    Average 82 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    i t4 - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    79
    TaKaRa t4 dcdna
    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by <t>T4</t> RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.
    T4 Dcdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dcdna/product/TaKaRa
    Average 79 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    t4 dcdna - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    Image Search Results


    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Journal: Nature Communications

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    doi: 10.1038/s41467-017-00484-w

    Figure Lengend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Article Snippet: Samples were treated with CIP or T4 PNK by addition of “Cutsmart” or “PNK” buffer from New England Biolabs and 10 units of CIP or T4 PNK and incubation at 37 °C for 15 min. Mock treated samples contained only Cutsmart buffer and water in lieu of CIP or T4 PNK.

    Techniques: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Journal: Aging and Disease

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

    doi: 10.14336/AD.2016.0910

    Figure Lengend Snippet: In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Article Snippet: Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Techniques: In Vitro, Purification, Activity Assay, Staining, Western Blot, Positive Control

    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Journal: Nature Communications

    Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions

    doi: 10.1038/s41467-019-09146-5

    Figure Lengend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Article Snippet: We confirmed the existence of these adducts by the sensitivity of this damaged template to an enzyme called, T4-PDG (from bacteriophage T4, pyrimidine dimer glycosylase; NEB) (Fig. , third lane).

    Techniques: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling

    Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), RPA-T4 ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.

    Journal: PLoS ONE

    Article Title: Thermal Stability of the Human Immunodeficiency Virus Type 1 (HIV-1) Receptors, CD4 and CXCR4, Reconstituted in Proteoliposomes

    doi: 10.1371/journal.pone.0013249

    Figure Lengend Snippet: Binding of conformation-dependent monoclonal antibodies against CD4 and CXCR4 to CD4/CXCR4-proteoliposomes. The indicated concentrations of PE-labeled monoclonal antibodies Q4120 ( A ), RPA-T4 ( B ), 12G5 ( C ) and 44717.111 ( D ) were incubated with CD4/CXCR4-proteoliposomes. The mean fluorescence intensity at each antibody concentration was normalized to that seen at the highest antibody concentration used. Each data point represents the mean and standard error derived from three independent experiments, each using a different preparation of CD4/CXCR4-proteoliposomes.

    Article Snippet: PE-labeled anti-human CCR5, clone 2D7; PE-labeled anti-human CXCR4, clone 12G5; and PE-labeled anti-human CD4, clone RPA-T4 were purchased from BD Biosciences Pharmingen (San Jose, CA).

    Techniques: Binding Assay, Labeling, Recombinase Polymerase Amplification, Incubation, Fluorescence, Concentration Assay, Derivative Assay

    Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs RPA-T4, SK3 and #21, or mouse IgG as

    Journal:

    Article Title: Interaction with CD4 and Antibodies to CD4-Induced Epitopes of the Envelope gp120 from a Microglial Cell-Adapted Human Immunodeficiency Virus Type 1 Isolate

    doi: 10.1128/JVI.79.11.6703-6713.2005

    Figure Lengend Snippet: Inhibition of pseudotype infection by anti-CD4 MAbs. Bori and Bori-15 envelope-pseudotyped luciferase viruses were used in single-round infection assays in the presence of increasing concentrations of anti-CD4 MAbs RPA-T4, SK3 and #21, or mouse IgG as

    Article Snippet: HOS cells stably expressing CD4 and CCR5 (plated at 104 cells/well into 96-well plates) were incubated for 1 h at 4°C with medium alone or in medium with anti-CD4 MAbs #21, RPA-T4 (eBioscience, San Diego, CA), SK3 (BD Biosciences), or mouse IgG and infected with pseudotypes for 6 h. Luciferase activity was measured at 2 to 3 days postinfection.

    Techniques: Inhibition, Infection, Luciferase, Recombinase Polymerase Amplification

    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Journal: Nucleic Acids Research

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    doi: 10.1093/nar/gkl449

    Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR.

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Journal: The Journal of Biological Chemistry

    Article Title: UV Damage in DNA Promotes Nucleosome Unwrapping *

    doi: 10.1074/jbc.M110.140087

    Figure Lengend Snippet: UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Article Snippet: Briefly, samples were incubated with 2.5 units of T4 DNA polymerase-exonuclease (Fermentas) at 37 °C for 2 h. The reaction was stopped by heating at 65 °C for 10 min.

    Techniques: Sequencing, Labeling, Concentration Assay, Staining, Southern Blot, Irradiation

    DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) T4 DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.

    Journal: Journal of Nucleic Acids

    Article Title: Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

    doi: 10.4061/2010/823917

    Figure Lengend Snippet: DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) T4 DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.

    Article Snippet: Materials T4 DNA ligase (10 U/μ L) was purchased from Invitrogen (Carlsbad, CA.).

    Techniques: Non-Homologous End Joining, Western Blot, Activity Assay, Standard Deviation, Positive Control, Negative Control

    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: DNA Ligation, Labeling, Incubation, Ligation, Electrophoresis

    Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Labeling, Incubation, Titration, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Incubation, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Ligation, Electrophoresis

    Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.

    Journal: Scientific Reports

    Article Title: Enzyme-guided DNA Sewing Architecture

    doi: 10.1038/srep17722

    Figure Lengend Snippet: Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.

    Article Snippet: To construct T-DNA, sequences were formed via a complementary hybridization of each base, followed by T4 ligase (Promega, Madison, WI).

    Techniques: Ligation, Activity Assay, Sequencing, Concentration Assay

    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Journal: Nature Communications

    Article Title: Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    doi: 10.1038/ncomms9430

    Figure Lengend Snippet: Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Article Snippet: To measure the half-life of saiRNA with terminal 2′, 3′-cyclic phosphate or hydroxyl groups, T7-transcribed saiRNA-RZs treated or untreated with T4 PNK were incubated with Ago2-KO 293 cell lysates in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 1% NP-40, 0.5% sodium deoxycholate (Sigma), 0.1 U μl−1 RNase Inhibitor and 1/100 protease inhibitor cocktail) for different time periods.

    Techniques: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, shRNA, Luciferase, Northern Blot, Western Blot, Transfection, Activity Assay, Stable Transfection, Transduction

    ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by T4 RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system

    doi: 10.1093/nar/gkl087

    Figure Lengend Snippet: ( A ) Schematic illustration of the formation of streptavidin–tRNA fusion using puromycin–tRNA, which contains a puromycin moiety in the place of 3′ terminal aminoacyl-adenosine and a four-base anticodon CCCG. The puromycin–tRNA binds to ribosomal A site and accepts a streptavidin polypeptide chain as an analog of aminoacyl-tRNA in response to a four-base CGGG codon at 3′ terminus of streptavidin mRNA in a cell-free translation. The resulting streptavidin–puromycin–tRNA may be translocated to the P-site. In this case, the next aminoacyl-tRNA binds to the vacant ribosomal A site, but can not accept the polypeptide chain because of the amide bond of puromycin–tRNA. The resulting streptavidin–tRNA fusion is released from the ribosome complex by the addition of EDTA. ( B ) Schematic illustration of the in vitro selection system of tRNAs. Step 1, a DNA pool encoding tRNAs containing a four-base anticodon CCCG is transcribed by T7 RNA polymerase to tRNA(-CA) pool. Step 2, the tRNA(-CA) pool is ligated with pdCp-Puromycin by T4 RNA ligase to generate puromycin–tRNA. Step 3, a streptavidin mRNA containing a four-base CGGG codon at C-terminus is translated in an E.coli cell-free translation system in the presence of the puromycin–tRNA. Puromycin–tRNAs that successfully decode the CGGG codon form ribosome–mRNA–streptavidin–tRNA complex. Step 4, the streptavidin–tRNA fusion is dissociated from the complex by the addition of EDTA. Step 5, the streptavidin–tRNA fusion is recovered with biotin-coated magnetic beads. Step 6, the streptavidin–tRNA fusion is dissociated from the beads, and then the tRNA moiety is subjected to RT–PCR. Step 7, the tRNA genes are regenerated by overlap-extension PCR with a T7 promoter primer, which are used as template DNAs in the next round of selection.

    Article Snippet: T4 RNA ligase, Bca BEST RNA PCR kit ver1.1, GelStar Nucleic Acid Stain and ribonuclease inhibitor were from TaKaRa BIO.

    Techniques: In Vitro, Selection, Magnetic Beads, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction