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  • 99
    New England Biolabs psti psti
    SILAC-based affinity capture of Cj1132c with purF <t>DNA</t> bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with <t>PstI</t> restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.
    Psti Psti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher psti fastdigest
    SILAC-based affinity capture of Cj1132c with purF <t>DNA</t> bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with <t>PstI</t> restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.
    Psti Fastdigest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc anti spink3
    Interaction of Htt with <t>Spink3</t> regulates trypsin activity in vitro. ( A ) Measurement of trypsin activity in vitro by adding Spink3, HTT, nHTT, tHTT, or Spink3 and HTT, nHTT, and tHTT containing cell lysates of transfected HEK293 cells. Cell extracts were mixed with porcine trypsin (TRSEQII) and the substrate Boc-Gln-Ala-Arg-AMC, and the rates of fluorescent products generated by trypsin were measured. Results are indicated as means ± SEM ( n = 3; two-way ANOVA test, * P
    Anti Spink3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher psti
    Circularization of <t>pBR322</t> (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by <t>PstI.</t> Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.
    Psti, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher fastdigest psti
    Circularization of <t>pBR322</t> (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by <t>PstI.</t> Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.
    Fastdigest Psti, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher xbai psti
    Circularization of <t>pBR322</t> (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by <t>PstI.</t> Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.
    Xbai Psti, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs psti hf
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Psti Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore psti enzyme
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Psti Enzyme, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega psti enzyme
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Psti Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher psti xhoi
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Psti Xhoi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa psti enzyme
    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Psti Enzyme, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.

    Journal: mBio

    Article Title: High-Frequency Variation of Purine Biosynthesis Genes Is a Mechanism of Success in Campylobacter jejuni

    doi: 10.1128/mBio.00612-15

    Figure Lengend Snippet: SILAC-based affinity capture of Cj1132c with purF DNA bait and effect of cj1132c deletion on spontaneous mutation rate. (A) Screen for DNA-binding proteins captured with purF DNA bait. Differentially labeled (light, [ 13 C 6 ]arginine, or heavy, [ 13 C 6 14 N 4 ]arginine) C. jejuni total protein lysates were incubated with biotinylated purF DNA or prsA DNA, respectively. A 1:1 light/heavy mixture of DNA/protein complexes was captured on streptavidin beads. Proteins were eluted from beads by digestion with PstI restriction enzyme, and trypsinized proteins were analyzed by gel electrophoresis liquid chromatography-mass spectrometry. Each dot represents an identified peptide. SILAC analysis discriminates the specificity of an interaction; peptides with a low heavy/light ratio indicate higher-affinity capture. Data are representative of two independent experiments statistically assessed by the Benjamini-Hochberg false discovery rate (FDR), and FDR Q significance values are indicated: *, Q = 0.024; **, Q = 0.014. (B) Representative mass spectra for SILAC-labeled captured peptides. Top, equal mass/charge ratio of light and heavy forms of PorA peptide indicating nonspecific interaction; bottom, the light form of Cj1132c is present in a ratio > 16.8 times higher than the heavy form, indicating a high probability of interaction with the direct repeat and hypervariable region of purF . (C) Frequency of emergence of spontaneous ciprofloxacin resistance (spontaneous mutation rate) of Δ cj1132c and complemented Δ cj1132c / cj1132c mutants in purF- T91del genetic background. The numbers of spontaneous ciprofloxacin-resistant mutants relative to total CFU per OD 600 equivalent are presented. Mean results with SEM from six independent experiments each with two technical replicates are shown: ****, P ≤ 0.0001.

    Article Snippet: Protein-DNA complexes were pooled, eluted by PstI (NEB) restriction enzyme cleavage, and digested with ArgC, and purified peptides were analyzed by reverse-phase liquid chromatography-mass spectrometry.

    Techniques: Mutagenesis, DNA Binding Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Liquid Chromatography, Mass Spectrometry

    Changes in conformational states of the terminase and prohead portal during packaging of linear and Y-DNA substrates. Packaging was carried out using GFP-gp20 proheads, CT-ReAsH terminase, and unlabeled linear (5-kb PstI-digested pL16 DNA) or unlabeled 3.7-kb Y-DNA. Different FRET values were observed between CT-ReAsH terminase and GFP-gp20 fusion with the linear ( A ) and Y-DNAs ( B ). FRET was measured with the 3.7-kb Y-DNA after resolvase treatment for 30 min ( C ).

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamics of the T4 Bacteriophage DNA Packasome Motor

    doi: 10.1074/jbc.M111.222828

    Figure Lengend Snippet: Changes in conformational states of the terminase and prohead portal during packaging of linear and Y-DNA substrates. Packaging was carried out using GFP-gp20 proheads, CT-ReAsH terminase, and unlabeled linear (5-kb PstI-digested pL16 DNA) or unlabeled 3.7-kb Y-DNA. Different FRET values were observed between CT-ReAsH terminase and GFP-gp20 fusion with the linear ( A ) and Y-DNAs ( B ). FRET was measured with the 3.7-kb Y-DNA after resolvase treatment for 30 min ( C ).

    Article Snippet: The double dye AxC3Y-DNAs, single dye AxY-DNAs, and unlabeled Y-DNAs (90 bases each) were phosphorylated by T4 polynucleotide kinase (New England Biolabs), and the blunt ends were ligated to pBR322 DNA linearized with EcoRV, followed by PstI digestion and gel extraction to yield the Y-DNAs with the 3.7-kb purified pBR322 (New England Biolabs) derived leaders ( C ).

    Techniques:

    ReAsH-EDT2 labeled N- and C-terminal tetra cysteine-tagged gp17 proteins are active in DNA packaging. A and B , both the NT and CT tetra cysteine-tagged gp17 proteins were labeled with ReAsH EDT2 dye, and fluorescence was measured in a Picoquant MicroTime 200 confocal microscope ( A ) and Typhoon imager ( B ). C , the labeled and unlabeled CT and NT terminases were run on native-PAGE followed by Coomassie staining as a loading control, and unstained gel was analyzed in Typhoon imager. D and E , nuclease assays were carried out to check the activities of the unlabeled ( D ) and the labeled CT and NT terminases ( E ) using 5-kb PstI linearized pL16 DNA as a substrate and wild type proheads in the reaction mixtures. Wild type terminase was used as a positive control in the assays.

    Journal: The Journal of Biological Chemistry

    Article Title: Dynamics of the T4 Bacteriophage DNA Packasome Motor

    doi: 10.1074/jbc.M111.222828

    Figure Lengend Snippet: ReAsH-EDT2 labeled N- and C-terminal tetra cysteine-tagged gp17 proteins are active in DNA packaging. A and B , both the NT and CT tetra cysteine-tagged gp17 proteins were labeled with ReAsH EDT2 dye, and fluorescence was measured in a Picoquant MicroTime 200 confocal microscope ( A ) and Typhoon imager ( B ). C , the labeled and unlabeled CT and NT terminases were run on native-PAGE followed by Coomassie staining as a loading control, and unstained gel was analyzed in Typhoon imager. D and E , nuclease assays were carried out to check the activities of the unlabeled ( D ) and the labeled CT and NT terminases ( E ) using 5-kb PstI linearized pL16 DNA as a substrate and wild type proheads in the reaction mixtures. Wild type terminase was used as a positive control in the assays.

    Article Snippet: The double dye AxC3Y-DNAs, single dye AxY-DNAs, and unlabeled Y-DNAs (90 bases each) were phosphorylated by T4 polynucleotide kinase (New England Biolabs), and the blunt ends were ligated to pBR322 DNA linearized with EcoRV, followed by PstI digestion and gel extraction to yield the Y-DNAs with the 3.7-kb purified pBR322 (New England Biolabs) derived leaders ( C ).

    Techniques: Labeling, Fluorescence, Microscopy, Clear Native PAGE, Staining, Positive Control

    Pre- and posttreatment of endogenous reactions with DEAE-RT preparations. Reaction mixtures were preincubated in standard endogenous reaction buffer with or without actinomycin D (Ac) or RNase A (R) for 5 min prior to the addition of [ 32 P]dCTP. (Left panel) Reaction products were treated with RNase A (R) or DNase I (D) or left untreated prior to precipitation and electrophoresis in a 1.5% nondenaturing agarose gel. (Middle panel) Endogenous reaction mixtures were subjected to the following treatments: lane 6, mock incubation; lane 7, incubation in 0.1 M NaOH; lane 8, incubation with proteinase K; lane 9, incubation with proteinase K and then extraction with phenol-CIA; lane 10, mock incubation, followed by extraction with phenol-CIA. Reaction products were treated with glyoxal prior to electrophoresis. (Right panel) Endogenous reaction products were incubated with (lane 12) or without (lane 11) λ exonuclease. Reaction products were treated with glyoxal prior to electrophoresis. The filled arrows indicate the 1.95-kb cDNA reaction product, and the unfilled arrow indicates a band which is detected in nondenaturing gels and is more pronounced in reactions that retain endogenous RNA. The size of the products was determined by 5′-end-labeled λ-PstI and λ-HindIII/EcoRI restriction fragment markers (data not shown).

    Journal: Eukaryotic Cell

    Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum

    doi: 10.1128/EC.3.6.1589-1600.2004

    Figure Lengend Snippet: Pre- and posttreatment of endogenous reactions with DEAE-RT preparations. Reaction mixtures were preincubated in standard endogenous reaction buffer with or without actinomycin D (Ac) or RNase A (R) for 5 min prior to the addition of [ 32 P]dCTP. (Left panel) Reaction products were treated with RNase A (R) or DNase I (D) or left untreated prior to precipitation and electrophoresis in a 1.5% nondenaturing agarose gel. (Middle panel) Endogenous reaction mixtures were subjected to the following treatments: lane 6, mock incubation; lane 7, incubation in 0.1 M NaOH; lane 8, incubation with proteinase K; lane 9, incubation with proteinase K and then extraction with phenol-CIA; lane 10, mock incubation, followed by extraction with phenol-CIA. Reaction products were treated with glyoxal prior to electrophoresis. (Right panel) Endogenous reaction products were incubated with (lane 12) or without (lane 11) λ exonuclease. Reaction products were treated with glyoxal prior to electrophoresis. The filled arrows indicate the 1.95-kb cDNA reaction product, and the unfilled arrow indicates a band which is detected in nondenaturing gels and is more pronounced in reactions that retain endogenous RNA. The size of the products was determined by 5′-end-labeled λ-PstI and λ-HindIII/EcoRI restriction fragment markers (data not shown).

    Article Snippet: DNA standards were dephosphorylated PstI restriction fragments of bacteriophage λ that had been 5′ end labeled with [γ-32 P]ATP by using polynucleotide kinase (New England Biolabs, Beverly, Mass.).

    Techniques: Electrophoresis, Agarose Gel Electrophoresis, Incubation, Labeling

    Interaction of Htt with Spink3 regulates trypsin activity in vitro. ( A ) Measurement of trypsin activity in vitro by adding Spink3, HTT, nHTT, tHTT, or Spink3 and HTT, nHTT, and tHTT containing cell lysates of transfected HEK293 cells. Cell extracts were mixed with porcine trypsin (TRSEQII) and the substrate Boc-Gln-Ala-Arg-AMC, and the rates of fluorescent products generated by trypsin were measured. Results are indicated as means ± SEM ( n = 3; two-way ANOVA test, * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

    doi: 10.1073/pnas.1524575113

    Figure Lengend Snippet: Interaction of Htt with Spink3 regulates trypsin activity in vitro. ( A ) Measurement of trypsin activity in vitro by adding Spink3, HTT, nHTT, tHTT, or Spink3 and HTT, nHTT, and tHTT containing cell lysates of transfected HEK293 cells. Cell extracts were mixed with porcine trypsin (TRSEQII) and the substrate Boc-Gln-Ala-Arg-AMC, and the rates of fluorescent products generated by trypsin were measured. Results are indicated as means ± SEM ( n = 3; two-way ANOVA test, * P

    Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

    Techniques: Activity Assay, In Vitro, Transfection, Generated

    Interaction of Htt with Spink3 inhibits trypsin activity. ( A ) Western blotting ( Upper ) and quantification ( Lower ) of Spink3 in 2-, 4-, and 8-mo-old ubiquitous KO and control mice. ( n = 3 independent experiments, two-way ANOVA test; n.s. represents no significant difference, *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

    doi: 10.1073/pnas.1524575113

    Figure Lengend Snippet: Interaction of Htt with Spink3 inhibits trypsin activity. ( A ) Western blotting ( Upper ) and quantification ( Lower ) of Spink3 in 2-, 4-, and 8-mo-old ubiquitous KO and control mice. ( n = 3 independent experiments, two-way ANOVA test; n.s. represents no significant difference, *** P

    Article Snippet: Rabbit antibodies used in this study were obtained from commercial sources as follows: HTT [EPR5526] (Abcam), beta-tubulin III [EP1569Y] (Novus Biologicals), NF-κB [D14E12] (Cell Signaling), LC3I/II [NB100-2220] (Novus Biologicals), ELA3B [GTX105123] (GeneTex), FAT10 [EPR4370] (GeneTex), SPINK3 [2744S] (Cell Signaling), HA [C29F4] (Cell Signaling), p-NF-κB [93H1] (Cell Signaling), RIP3 [GTX107574] (GeneTex), Caspase3 [9662] (Cell Signaling), and cleaved Caspase3 [5A1E] (Cell Signaling).

    Techniques: Activity Assay, Western Blot, Mouse Assay

    Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.

    Journal: PLoS ONE

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    doi: 10.1371/journal.pone.0177751

    Figure Lengend Snippet: Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.

    Article Snippet: Electrophoretic Mobility Shift Assay (EMSA) 200 ng of supercoiled pBR322 or pBR322 linearized by PstI (Thermo-Scientific) (4361 bp), as well as 200 ng of RFI (5386 bp) or single-stranded DNA of phiX174 virion were incubated in 20 μl of buffer A (40 mM Tris-HCl pH 7.8, 5 mM MgCl2 , 1.5 mM DTT, 50 mM NaCl, 12% glycerol) with increasing concentrations of DdrC (0.86 μM, 1.7 μM, 3.5 μM, 7 μM and 8.6 μM).

    Techniques: Electron Microscopy, Plasmid Preparation

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation