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  • 95
    Millipore dna fragments
    Comparative evaluation of the anti-apoptotic function of <t>aFGF28-154</t> both in vitro and in vivo . A: TEM observation (× 20000) of DEX-induced apoptosis in mouse thymocytes. B: Typical apoptotic bodies in DEX-induced thymocytes. C: Damage of <t>DNA</t> analysis by agarose gel electrophoresis according to DNA ladder kit (Apoptosis DNA Laddering Kit-Ethidium Bromide, R D Systems). Lane 1: DNA extracted from normal control group; lane 2: DNA from DEX only group; lane 3: DNA from DEX plus aFGF28-154 group; lane 4: DNA from DEX plus aFGF group; lane 5: DNA size markers (bp). D: Flow cytometry results.
    Dna Fragments, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 4639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher bp dna fragment
    <t>DNA</t> capture and sequencing with NZMWs a. Schematic of capture a nd activation of a DNA/DNA polymerase-streptavidin fusion complex in an NZMW: voltage is used to draw a 72-nucleotide circular DNA/polymerase complex into the sequencing volume of a biotinylated NZMW and bind it to the surface (left). Polymerase activation by adding Mg 2+ initiates sequencing, which results in discrete fluorescence bursts from the NZMW. b. Top: Fluorescence images of a 2 × 2 NZMW array under 640 nm illumination at several points during a proof-of-principle experiment. NZMWs are bounded by the red box (note: prism not used for acquiring these images). Bottom: extracted fluorescence trace from the top left NZMW in the above images. Roman numerals correspond to the points identified in the top images. i . V = 0 mV, template DNA and dNTP analogues in solution. ii . V = 750 mV, template DNA and dNTP analogues are drawn into NZMWs. iii . V = 0 mV, before Mg 2+ addition. iv . V = 0 mV, after Mg 2+ addition (shown at different times). v . V = 0 mV, after KCl spike to final concentration of 850 mM (also see SI Movie 1 ). c. Excerpts (1.1 s long) showing successive frames (left to right) during sequencing of a 20 kbp <t>SMRTbell</t> template. Numbers to left correspond to initial time of excerpt from an acquired movie. The first instance of each of the four nucleotide incorporations shown in the excerpts is labelled using coloured letters. d. False-colour spectrally-resolved fluorescence intensity vs. time data obtained during a 20 kbp sequencing experiment (see main text and SI Movie 2 for raw data). Legend shows the colour of each of the dNTPs (see SI, Section 10 for alignment of the 1.6 kbp read to the 20 kbp template).
    Bp Dna Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Roche deoxyribonucleic acid dna fragments
    Microglial responses to different doses of LPS. ( a ) Low dose of LPS increased microglia viability whereas higher doses triggered cell death. Primary rat microglia were stimulated with increasing concentrations of LPS (0.1, 10, 100 ng/ml) for 20–24 h, and the number of microglial cells were counted. Data are a representative of three independent experiments with similar results. ( b ) Representative phase-contrast images of microglia treated with indicated concentrations of LPS. Scale bar, 50 μ m. ( c ) Significant increase of <t>TUNEL-positive</t> microglia after LPS stimulation for 6 h. Data represent the percentage of TUNEL-positive cells of the total cells. ( d ) Dose-dependent increase of TNF production upon LPS treatment for 6 h. Data are a representative of three independent experiments with similar results. ( e , f ) LPS-activated microglia exhibited two distinct casaspe-8 activation patterns. Localized caspase-8 activation (L-Casp-8) was not associated with caspase-3 activation or cell death (upper panel, e ), whereas profound cytoplasmic activation of caspase-8 (C-Casp-8) induced caspase-3 activation, <t>DNA</t> fragmentation (TUNEL-positive) and cell death (lower panel, e ). Rat microglia were treated with 0.1–100 ng/ml LPS as above and in situ detection of caspase-8 activation in live cells was carried out as described in Methods section. Cells were then fixed and subjected to TUNEL labeling or immunostaining for cleaved/activated caspase-3 (act. Casp-3). Results are shown as percentage of Casp-8-positive cells of total cells. NS, not significant; * P
    Deoxyribonucleic Acid Dna Fragments, supplied by Roche, used in various techniques. Bioz Stars score: 76/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega dna fragments
    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were <t>PCR</t> amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual <t>DNA</t> fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.
    Dna Fragments, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 8246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna fragments
    <t>DNA</t> sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File <t>S1</t> .
    Dna Fragments, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GenScript dna fragments
    <t>DNA</t> sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File <t>S1</t> .
    Dna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 94/100, based on 407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATUM dna fragments
    <t>DNA</t> sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File <t>S1</t> .
    Dna Fragments, supplied by ATUM, used in various techniques. Bioz Stars score: 94/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Blue Heron Biotech dna fragments
    <t>DNA</t> sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File <t>S1</t> .
    Dna Fragments, supplied by Blue Heron Biotech, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cambrex dna fragments
    <t>DNA</t> sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File <t>S1</t> .
    Dna Fragments, supplied by Cambrex, used in various techniques. Bioz Stars score: 90/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Illumina Inc dna fragments
    Preparation of paired-end reduced representation libraries. Genomic <t>DNA</t> is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large <t>contig</t> sequences.
    Dna Fragments, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 95/100, based on 6938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    MWG-Biotech dna fragment
    Preparation of paired-end reduced representation libraries. Genomic <t>DNA</t> is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large <t>contig</t> sequences.
    Dna Fragment, supplied by MWG-Biotech, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    APELEX dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by APELEX, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 5417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Genewiz dna fragment
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragment, supplied by Genewiz, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Twist Bioscience dna fragments
    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with <t>ethidium</t> bromide for determination –108C > T polymorphism. <t>DNA</t> ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.
    Dna Fragments, supplied by Twist Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    CEM Corporation dna fragments
    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear <t>DNA</t> cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two <t>CEM-T4</t> clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.
    Dna Fragments, supplied by CEM Corporation, used in various techniques. Bioz Stars score: 90/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Kapa Biosystems dna fragments
    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear <t>DNA</t> cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two <t>CEM-T4</t> clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.
    Dna Fragments, supplied by Kapa Biosystems, used in various techniques. Bioz Stars score: 94/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Novogene dna fragment
    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear <t>DNA</t> cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two <t>CEM-T4</t> clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.
    Dna Fragment, supplied by Novogene, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Roche dna fragments
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    Cosmo Genetech Co dna fragments
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    91
    TaKaRa dna fragment
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
    Dna Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    ATUM pftsn dna fragment
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    Eurofins ndei dna fragment
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    Illumina Inc target dna fragments
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    Thermo Fisher dna fragment analysis
    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated <t>DIG-labeled</t> <t>DNA</t> probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).
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    Thermo Fisher string dna fragment
    <t>αS</t> inclusion dynamics. ( A ) Schematics of wt αS and repeat-motif mutants by aligning aa sequences via the repeat consensus sequence KTKEGV. Middle and right panels: immunofluorescent images (YFP, green; DAPI, blue), live cells, 5 and 20 days after transfection of M17D neuroblastoma cells with the indicated αS::YFP variants followed by Zeocin selection. Scale bar, 20 µm. ( B ) wt vs. 3K, and wt vs. KLK αS were transiently expressed in M17D cells for 48 h followed by DSG intact-cell cross-linking and Western blot (WB). As a control, mock-transfected cells (-) are shown. Two different <t>DNA</t> amounts (4 and 8 µg per 6-cm dish, respectively) were transfected in the case of wt vs. 3K (left panels), total protein lysate and blots were developed for αS (mAb Syn-1), DJ-1 (dimeric protein, positive control/loading control) and Ran (monomeric protein, negative control/loading control). Wt vs. KLK (right panels, mAb 15G7) was analyzed by sequential extraction (PBS fraction, ∼cytosol; TX-100 fraction) and adding DSP/βME-reversed crosslinking (see Methods) to visualize total αS amounts. Note that, consistent with earlier data, we sometimes observe αS30 putative dimers upon transient overexpression, while apparent trimers are typically absent. Data represent at least 10 independent experiments. Membranes were cut as indicated by thin white lines. ( C ) Time-course of αS-wt::YFP induction in a clonal cell line. M17D cells expressing YFP-tagged αS 3K (dox-inducible) were induced for the indicated time points (all cells plated at the same time, but induced at different time points), then fixed (4% paraformaldehyde) and imaged. Only very few smaller inclusions (
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    Thermo Fisher strings dna fragments
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Corbett Life Science dna fragment analyser
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    GenScript genparts dna fragment
    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with <t>DNA</t> encoding <t>SthK-bP</t> and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .
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    Image Search Results


    Comparative evaluation of the anti-apoptotic function of aFGF28-154 both in vitro and in vivo . A: TEM observation (× 20000) of DEX-induced apoptosis in mouse thymocytes. B: Typical apoptotic bodies in DEX-induced thymocytes. C: Damage of DNA analysis by agarose gel electrophoresis according to DNA ladder kit (Apoptosis DNA Laddering Kit-Ethidium Bromide, R D Systems). Lane 1: DNA extracted from normal control group; lane 2: DNA from DEX only group; lane 3: DNA from DEX plus aFGF28-154 group; lane 4: DNA from DEX plus aFGF group; lane 5: DNA size markers (bp). D: Flow cytometry results.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Enhanced anti-apoptosis and gut epithelium protection function of acidic fibroblast growth factor after cancelling of its mitogenic activity

    doi: 10.3748/wjg.v10.i24.3590

    Figure Lengend Snippet: Comparative evaluation of the anti-apoptotic function of aFGF28-154 both in vitro and in vivo . A: TEM observation (× 20000) of DEX-induced apoptosis in mouse thymocytes. B: Typical apoptotic bodies in DEX-induced thymocytes. C: Damage of DNA analysis by agarose gel electrophoresis according to DNA ladder kit (Apoptosis DNA Laddering Kit-Ethidium Bromide, R D Systems). Lane 1: DNA extracted from normal control group; lane 2: DNA from DEX only group; lane 3: DNA from DEX plus aFGF28-154 group; lane 4: DNA from DEX plus aFGF group; lane 5: DNA size markers (bp). D: Flow cytometry results.

    Article Snippet: Plasmids encoding aFGF28-154 were generated by amplification of appropriate DNA fragments, followed by subcloning the products into pET-3c vectors. aFGF28-154 protein was expressed in BL21 (DE3) cells and purified on an M2 agarose affinity column (Sigma).

    Techniques: In Vitro, In Vivo, Transmission Electron Microscopy, Agarose Gel Electrophoresis, DNA Laddering, Flow Cytometry, Cytometry

    Morphological examination and immunohistochemical detection and quantification of apoptosis based on the label-ling of DNA strand breaks (Roche Applied Science, Germany). A: Marked epithelial separation from the basement, subepithelial edema, haemorrhage, erosion. C: Necrosis in I/R plus saline control group. E and G: Tissue damage reduction in both aFGF and aFGF28-154 treated groups. B: Immunohistochemical detection and quantification of apoptosis in normal epithelium entericum. D: Increased number of apoptotic cells in epithelium entericum in rats treated with saline. F and H: Significant reduction of apoptotic cells in epithelium entericum in rats treated with both aFGF and aFGF28-154.

    Journal: World Journal of Gastroenterology : WJG

    Article Title: Enhanced anti-apoptosis and gut epithelium protection function of acidic fibroblast growth factor after cancelling of its mitogenic activity

    doi: 10.3748/wjg.v10.i24.3590

    Figure Lengend Snippet: Morphological examination and immunohistochemical detection and quantification of apoptosis based on the label-ling of DNA strand breaks (Roche Applied Science, Germany). A: Marked epithelial separation from the basement, subepithelial edema, haemorrhage, erosion. C: Necrosis in I/R plus saline control group. E and G: Tissue damage reduction in both aFGF and aFGF28-154 treated groups. B: Immunohistochemical detection and quantification of apoptosis in normal epithelium entericum. D: Increased number of apoptotic cells in epithelium entericum in rats treated with saline. F and H: Significant reduction of apoptotic cells in epithelium entericum in rats treated with both aFGF and aFGF28-154.

    Article Snippet: Plasmids encoding aFGF28-154 were generated by amplification of appropriate DNA fragments, followed by subcloning the products into pET-3c vectors. aFGF28-154 protein was expressed in BL21 (DE3) cells and purified on an M2 agarose affinity column (Sigma).

    Techniques: Immunohistochemistry

    DNA capture and sequencing with NZMWs a. Schematic of capture a nd activation of a DNA/DNA polymerase-streptavidin fusion complex in an NZMW: voltage is used to draw a 72-nucleotide circular DNA/polymerase complex into the sequencing volume of a biotinylated NZMW and bind it to the surface (left). Polymerase activation by adding Mg 2+ initiates sequencing, which results in discrete fluorescence bursts from the NZMW. b. Top: Fluorescence images of a 2 × 2 NZMW array under 640 nm illumination at several points during a proof-of-principle experiment. NZMWs are bounded by the red box (note: prism not used for acquiring these images). Bottom: extracted fluorescence trace from the top left NZMW in the above images. Roman numerals correspond to the points identified in the top images. i . V = 0 mV, template DNA and dNTP analogues in solution. ii . V = 750 mV, template DNA and dNTP analogues are drawn into NZMWs. iii . V = 0 mV, before Mg 2+ addition. iv . V = 0 mV, after Mg 2+ addition (shown at different times). v . V = 0 mV, after KCl spike to final concentration of 850 mM (also see SI Movie 1 ). c. Excerpts (1.1 s long) showing successive frames (left to right) during sequencing of a 20 kbp SMRTbell template. Numbers to left correspond to initial time of excerpt from an acquired movie. The first instance of each of the four nucleotide incorporations shown in the excerpts is labelled using coloured letters. d. False-colour spectrally-resolved fluorescence intensity vs. time data obtained during a 20 kbp sequencing experiment (see main text and SI Movie 2 for raw data). Legend shows the colour of each of the dNTPs (see SI, Section 10 for alignment of the 1.6 kbp read to the 20 kbp template).

    Journal: Nature nanotechnology

    Article Title: Length-Independent DNA Packing into Nanopore Zero-Mode Waveguides for Low-Input DNA Sequencing

    doi: 10.1038/nnano.2017.176

    Figure Lengend Snippet: DNA capture and sequencing with NZMWs a. Schematic of capture a nd activation of a DNA/DNA polymerase-streptavidin fusion complex in an NZMW: voltage is used to draw a 72-nucleotide circular DNA/polymerase complex into the sequencing volume of a biotinylated NZMW and bind it to the surface (left). Polymerase activation by adding Mg 2+ initiates sequencing, which results in discrete fluorescence bursts from the NZMW. b. Top: Fluorescence images of a 2 × 2 NZMW array under 640 nm illumination at several points during a proof-of-principle experiment. NZMWs are bounded by the red box (note: prism not used for acquiring these images). Bottom: extracted fluorescence trace from the top left NZMW in the above images. Roman numerals correspond to the points identified in the top images. i . V = 0 mV, template DNA and dNTP analogues in solution. ii . V = 750 mV, template DNA and dNTP analogues are drawn into NZMWs. iii . V = 0 mV, before Mg 2+ addition. iv . V = 0 mV, after Mg 2+ addition (shown at different times). v . V = 0 mV, after KCl spike to final concentration of 850 mM (also see SI Movie 1 ). c. Excerpts (1.1 s long) showing successive frames (left to right) during sequencing of a 20 kbp SMRTbell template. Numbers to left correspond to initial time of excerpt from an acquired movie. The first instance of each of the four nucleotide incorporations shown in the excerpts is labelled using coloured letters. d. False-colour spectrally-resolved fluorescence intensity vs. time data obtained during a 20 kbp sequencing experiment (see main text and SI Movie 2 for raw data). Legend shows the colour of each of the dNTPs (see SI, Section 10 for alignment of the 1.6 kbp read to the 20 kbp template).

    Article Snippet: The 20kb SMRTbell template was prepared from NoLimits 20,000 bp DNA Fragment (Thermo Fisher Scientific, Inc.) using the SMRTbell template prep kit 1.0 (Pac Bio, Inc.) and size-selected with 15kb cut off using BluePippin instrument (Sage Science, Inc.).

    Techniques: Sequencing, Activation Assay, Fluorescence, Concentration Assay

    Microglial responses to different doses of LPS. ( a ) Low dose of LPS increased microglia viability whereas higher doses triggered cell death. Primary rat microglia were stimulated with increasing concentrations of LPS (0.1, 10, 100 ng/ml) for 20–24 h, and the number of microglial cells were counted. Data are a representative of three independent experiments with similar results. ( b ) Representative phase-contrast images of microglia treated with indicated concentrations of LPS. Scale bar, 50 μ m. ( c ) Significant increase of TUNEL-positive microglia after LPS stimulation for 6 h. Data represent the percentage of TUNEL-positive cells of the total cells. ( d ) Dose-dependent increase of TNF production upon LPS treatment for 6 h. Data are a representative of three independent experiments with similar results. ( e , f ) LPS-activated microglia exhibited two distinct casaspe-8 activation patterns. Localized caspase-8 activation (L-Casp-8) was not associated with caspase-3 activation or cell death (upper panel, e ), whereas profound cytoplasmic activation of caspase-8 (C-Casp-8) induced caspase-3 activation, DNA fragmentation (TUNEL-positive) and cell death (lower panel, e ). Rat microglia were treated with 0.1–100 ng/ml LPS as above and in situ detection of caspase-8 activation in live cells was carried out as described in Methods section. Cells were then fixed and subjected to TUNEL labeling or immunostaining for cleaved/activated caspase-3 (act. Casp-3). Results are shown as percentage of Casp-8-positive cells of total cells. NS, not significant; * P

    Journal: Cell Death & Disease

    Article Title: Caspase blockade induces RIP3-mediated programmed necrosis in Toll-like receptor-activated microglia

    doi: 10.1038/cddis.2013.238

    Figure Lengend Snippet: Microglial responses to different doses of LPS. ( a ) Low dose of LPS increased microglia viability whereas higher doses triggered cell death. Primary rat microglia were stimulated with increasing concentrations of LPS (0.1, 10, 100 ng/ml) for 20–24 h, and the number of microglial cells were counted. Data are a representative of three independent experiments with similar results. ( b ) Representative phase-contrast images of microglia treated with indicated concentrations of LPS. Scale bar, 50 μ m. ( c ) Significant increase of TUNEL-positive microglia after LPS stimulation for 6 h. Data represent the percentage of TUNEL-positive cells of the total cells. ( d ) Dose-dependent increase of TNF production upon LPS treatment for 6 h. Data are a representative of three independent experiments with similar results. ( e , f ) LPS-activated microglia exhibited two distinct casaspe-8 activation patterns. Localized caspase-8 activation (L-Casp-8) was not associated with caspase-3 activation or cell death (upper panel, e ), whereas profound cytoplasmic activation of caspase-8 (C-Casp-8) induced caspase-3 activation, DNA fragmentation (TUNEL-positive) and cell death (lower panel, e ). Rat microglia were treated with 0.1–100 ng/ml LPS as above and in situ detection of caspase-8 activation in live cells was carried out as described in Methods section. Cells were then fixed and subjected to TUNEL labeling or immunostaining for cleaved/activated caspase-3 (act. Casp-3). Results are shown as percentage of Casp-8-positive cells of total cells. NS, not significant; * P

    Article Snippet: To label fragmented DNA, TUNEL was performed using an in situ cell death detection kit (Roche, Indianapolis, IN, USA).

    Techniques: TUNEL Assay, Activation Assay, In Situ, Labeling, Immunostaining, Activated Clotting Time Assay

    EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were PCR amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual DNA fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.

    Journal: Journal of Bacteriology

    Article Title: Rex (Encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough Is a Repressor of Sulfate Adenylyl Transferase and Is Regulated by NADH

    doi: 10.1128/JB.02083-14

    Figure Lengend Snippet: EMSA demonstrating specific interaction between Rex DvH and the predicted Rex DvH -binding site within the sat promoter. (A) Schematic representation of the sat promoter region drawn approximately to scale. The predicted Rex DvH -binding site is annotated by an oval. Fragments (A, B, C, and D) used in EMSA are shown with their positions noted. Fragments A (121 bp), B (130 bp), and D (271 bp) were PCR amplified, while fragment C (40 bp) was generated by annealing two oligonucleotides. (B) Native polyacrylamide gel of individual DNA fragments (A, B, C, and D; 1 nM stock prior to column purification) without (−) or with (+) Rex DvH . An equal concentration of DNA was labeled and then passed over a column to separate the fragments from the rest of the components, i.e., unlabeled nucleotides. Fragment C, the smallest fragment, is below the size cutoff for the column (∼100 bp), and so only a small amount of this fragment is actually recovered compared to the other three larger fragments. Each fragment was eluted in the same volume of buffer, and so the concentration of this smaller fragment is considerably lower than the rest. Therefore, the band intensity for fragment C is less than the others. The lowest band common in all lanes is the dye front.

    Article Snippet: Genomic DNA (Wizard Genomic DNA purification kit; Promega, Madison, WI), plasmid (GeneJET plasmid kit; Thermo Fisher Scientific), and DNA fragments (Wizard SV Gel and PCR Clean-Up System [Promega]) were purified according to the manufacturer's protocol.

    Techniques: Binding Assay, Polymerase Chain Reaction, Amplification, Generated, Purification, Concentration Assay, Labeling

    DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Maize Transposable Elements Ac/Ds as Insertion Mutagenesis Tools in Candida albicans

    doi: 10.1534/g3.117.300388

    Figure Lengend Snippet: DNA sequences at the Dissociation ( Ds ) -NAT1 excision and reinsertion sites. (A) Ds excision footprints of 128 independent KMY103G1 ADE2 revertant colonies. The top row shows the ade2 :: Ds-NAT1 sequence on chromosome 3. Rows 1–28 show the recovered Ds excision footprints and their incidence ( F ) in independent ADE2 revertants from Ac TPase4xCa-expressing KMY103G1 cells grown in maltose-containing medium. Arrows above the sequences indicate inverted repeats centered around the complementary bases C and A (boldface red letters) of the nucleotides bordering the Ds-NAT1 transposon that result from the resolution of intermediate hairpin structures. Lower case letters indicate nucleotides that are not explained with the hairpin model. (B) Target site duplications at Ds-NAT1 reinsertion sites in transposants “1,” “2,” and “3”; see Table S2 in File S1 .

    Article Snippet: Construction of Ac/Ds components The codon-adapted Ac TPase4xCa open reading frame (Supplemental Material, Figure S1 in File S1 ) was constructed by fusing three de novo -synthesized DNA fragments (Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany) and cloned into pJET1.2 via the CloneJET kit (Thermo Fisher Scientific).

    Techniques: Sequencing, Expressing

    Intracellular expression of viral proteins of prM and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear DNA was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).

    Journal: Journal of Virology

    Article Title: Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly

    doi: 10.1128/JVI.00447-16

    Figure Lengend Snippet: Intracellular expression of viral proteins of prM and E TMD mutants. (A) Immunofluorescence staining of BHK cells transfected with mutant or WT viral RNAs, as indicated in the left panels. Staining was carried out using a polyclonal serum recognizing the prM/M and E proteins of TBEV and a monoclonal antibody recognizing NS1. Nuclear DNA was stained with Hoechst dye. The data are representative examples of three or more independent experiments. (B) Quantification of E in cell lysates by ELISA at 30 h posttransfection. (C) Quantification of NS1 in fixed cells by ELISA at 30 h posttransfection. The absorbance ratio of NS1 to GAPDH (cellular protein control) was determined, and the results are expressed as percentages of the ratio obtained with the WT. The data are presented as means ± standard errors for at least four independent experiments. Asterisks indicate significant differences relative to the WT (ANOVA and Dunnett's multiple-comparison test).

    Article Snippet: The membrane anchor region ( and ) of wild-type (WT) TBEV E [E(T1-T2)] or prM [prM(T1-T2)] in the pTNd/c vector was replaced by a chemically synthesized DNA fragment (GeneArt AG, Germany) containing the heterologous membrane anchor of JEV [E(J1-J2) or prM(J1-J2)] or a shuffled membrane anchor [E(J1-T2), E(T1-J2), prM(J1-T2), or prM(T1-J2)] by using unique restriction sites.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay

    DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC , diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB 53 version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region ( TRAC ) and a homologous sequence shared by both TCR β constant regions ( TRBC1/2 ) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification

    doi: 10.1016/j.omtm.2017.01.005

    Figure Lengend Snippet: DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC , diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB 53 version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region ( TRAC ) and a homologous sequence shared by both TCR β constant regions ( TRBC1/2 ) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.

    Article Snippet: Donor Construction and Targeted Integration PCR For the 200 bp α chain donor construct (TA200G), we ordered a GeneArt Strings DNA Fragment (Thermo Fisher Scientific) containing two 200 bp regions flanking the αT4 cutting site in the TRAC locus and restriction sites AsiSI and SbfI for cloning into a lentiviral transfer vector in antisense direction.

    Techniques: Non-Homologous End Joining, CRISPR, Gene Knockout, Expressing, Functional Assay, Knock-Out, Sequencing, Plasmid Preparation

    Preparation of paired-end reduced representation libraries. Genomic DNA is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large contig sequences.

    Journal: International Journal of Plant Genomics

    Article Title: Local Assemblies of Paired-End Reduced Representation Libraries Sequenced with the Illumina Genome Analyzer in Maize

    doi: 10.1155/2012/360598

    Figure Lengend Snippet: Preparation of paired-end reduced representation libraries. Genomic DNA is digested with methyl-sensitive restriction endonuclease PstI . After random shearing, DNA fragments containing the PstI end are selected via biotin selection and end-sequenced. Resulting sequences are assembled locally to create large contig sequences.

    Article Snippet: Contig length essentially varies with the size of the DNA fragments selected during the library construction procedure and is expected to be limited by the maximum length of the DNA fragments sequenced on the Illumina platform (separate experiments have shown that DNA fragments up to ~1 Kbps can be sequenced effectively on the Illumina Genome Analyzer).

    Techniques: Selection

    PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with ethidium bromide for determination –108C > T polymorphism. DNA ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.

    Journal: The Open Biochemistry Journal

    Article Title: Alzheimer’s Disease and Paraoxonase 1 (PON1) Gene Polymorphisms

    doi: 10.2174/1874091X01711010047

    Figure Lengend Snippet: PCR-restriction enzyme ( Bsr BI digestion) fragmentation patterns on the agarose gel stained with ethidium bromide for determination –108C > T polymorphism. DNA ladder (100bp) was loaded into well 1; wells 2 and 5 were CT; wells 3 and 7 were TT, wells 4 and 6 were CC.

    Article Snippet: Agarose gel (2%) stained with ethidium bromide (0.5 μg/ml) was used for electrophoresis of DNA fragments (Apelex, France).

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear DNA cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two CEM-T4 clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.

    Journal: PLoS ONE

    Article Title: Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs

    doi: 10.1371/journal.pone.0127986

    Figure Lengend Snippet: Nanocapsule delivery of gRNAs to excise the HIV-1 provirus by CRISPR mutagenesis. a) Schematic illustrating the position of two adjacent gRNAs directed to the HIV-1 LTR. b) A time course for knockout of EGFP expression was determined by flow cytometry. U6-control shRNA (CCR5-shRNA (a short linear DNA cassette with sh1005 shRNA expressed by U6 promoter)) was used a negative control. Dead cells were excluded by Live/Dead cell viability assay. c) Disruption of EGFP expression in two CEM-T4 clones, each bearing a single integrated EGFP lentiviral vector (FG11 EGFP). The U6-gRNA DNA cassette was encapsulated by nanocapsules, the hCas9 plasmid was condensed by PEI. The integration site of the lentiviral vector in Clone 1 is at chromosome (chr) 7 (-16350165). The integration site in Clone 2 is at chromosome (chr) 3 (+37026604). U6-control shRNA was used as a negative control. Dead cells were excluded by Live/Dead cell viability assay. Transduction efficiency is estimated to be 69.7% by transducing a Rhodamine B-labeled DNA cassette. d) EGFP expression after CRISPR/Cas9 nickase treatment. CEM-T4 cells were co-transduced with PEI condensed hCas9 nickase and gRNA nanocapsules. Dead cells were excluded by Live/Dead cell viability assay. e) Sequence analysis of the target site in the TAR region of LTR after the gRNA1/Cas9 treatment. DNA sequence demonstrated a single remaining LTR footprint resulting from proviral excision. The target sequence is indicated in red. The host cell genome sequence with integrated HIV vector is indicated as wild type (WT) on the top.

    Article Snippet: Edited DNA fragments from CEM Clone 1 were isolated using primer sets flanking the provirus indicated in .

    Techniques: CRISPR, Mutagenesis, Knock-Out, Expressing, Flow Cytometry, Cytometry, shRNA, Negative Control, Viability Assay, Clone Assay, Plasmid Preparation, Transduction, Labeling, Sequencing

    Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated DIG-labeled DNA probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).

    Journal: Journal of Bacteriology

    Article Title: Dual Role of LldR in Regulation of the lldPRD Operon, Involved in l-Lactate Metabolism in Escherichia coli

    doi: 10.1128/JB.02013-07

    Figure Lengend Snippet: Binding of LldR and PdhR to promoter fragments containing the O1 or O2 operator site. (A) (Left) Diagram of the lldP promoter region with the proposed O1 and O2 sites and the promoter fragments used as probes (P77 and P85). (Right) Sequence alignment of the PdhR operator present in the pdhR-aceEF-ldp operon promoter and the operators O1 and O2 in the lldP promoter. The arrows indicate the inverted repeat present in the operator sites recognized by GntR-like bacterial proteins. (B) Gel shift assays performed with the indicated DIG-labeled DNA probes. Probes P77 (encompassing O1) and P85 (encompassing O2) were added to binding mixtures containing 15 pmol of either purified LldR or PdhR. The probe encompassing the PdhR operator site was added to binding mixtures containing increasing amounts of PdhR (0.1, 0.4, 0.8, or 2 pmol). Reaction mixtures were incubated at 30°C for 15 min and directly subjected to polyacrylamide gel electrophoresis (PAGE).

    Article Snippet: A nonradioactive digoxigenin (DIG) gel shift kit for 3′-end labeling of DNA fragments (Roche Diagnostics, GmbH) was used for protein-DNA binding assays.

    Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Purification, Incubation, Polyacrylamide Gel Electrophoresis

    αS inclusion dynamics. ( A ) Schematics of wt αS and repeat-motif mutants by aligning aa sequences via the repeat consensus sequence KTKEGV. Middle and right panels: immunofluorescent images (YFP, green; DAPI, blue), live cells, 5 and 20 days after transfection of M17D neuroblastoma cells with the indicated αS::YFP variants followed by Zeocin selection. Scale bar, 20 µm. ( B ) wt vs. 3K, and wt vs. KLK αS were transiently expressed in M17D cells for 48 h followed by DSG intact-cell cross-linking and Western blot (WB). As a control, mock-transfected cells (-) are shown. Two different DNA amounts (4 and 8 µg per 6-cm dish, respectively) were transfected in the case of wt vs. 3K (left panels), total protein lysate and blots were developed for αS (mAb Syn-1), DJ-1 (dimeric protein, positive control/loading control) and Ran (monomeric protein, negative control/loading control). Wt vs. KLK (right panels, mAb 15G7) was analyzed by sequential extraction (PBS fraction, ∼cytosol; TX-100 fraction) and adding DSP/βME-reversed crosslinking (see Methods) to visualize total αS amounts. Note that, consistent with earlier data, we sometimes observe αS30 putative dimers upon transient overexpression, while apparent trimers are typically absent. Data represent at least 10 independent experiments. Membranes were cut as indicated by thin white lines. ( C ) Time-course of αS-wt::YFP induction in a clonal cell line. M17D cells expressing YFP-tagged αS 3K (dox-inducible) were induced for the indicated time points (all cells plated at the same time, but induced at different time points), then fixed (4% paraformaldehyde) and imaged. Only very few smaller inclusions (

    Journal: Human Molecular Genetics

    Article Title: Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells

    doi: 10.1093/hmg/ddx227

    Figure Lengend Snippet: αS inclusion dynamics. ( A ) Schematics of wt αS and repeat-motif mutants by aligning aa sequences via the repeat consensus sequence KTKEGV. Middle and right panels: immunofluorescent images (YFP, green; DAPI, blue), live cells, 5 and 20 days after transfection of M17D neuroblastoma cells with the indicated αS::YFP variants followed by Zeocin selection. Scale bar, 20 µm. ( B ) wt vs. 3K, and wt vs. KLK αS were transiently expressed in M17D cells for 48 h followed by DSG intact-cell cross-linking and Western blot (WB). As a control, mock-transfected cells (-) are shown. Two different DNA amounts (4 and 8 µg per 6-cm dish, respectively) were transfected in the case of wt vs. 3K (left panels), total protein lysate and blots were developed for αS (mAb Syn-1), DJ-1 (dimeric protein, positive control/loading control) and Ran (monomeric protein, negative control/loading control). Wt vs. KLK (right panels, mAb 15G7) was analyzed by sequential extraction (PBS fraction, ∼cytosol; TX-100 fraction) and adding DSP/βME-reversed crosslinking (see Methods) to visualize total αS amounts. Note that, consistent with earlier data, we sometimes observe αS30 putative dimers upon transient overexpression, while apparent trimers are typically absent. Data represent at least 10 independent experiments. Membranes were cut as indicated by thin white lines. ( C ) Time-course of αS-wt::YFP induction in a clonal cell line. M17D cells expressing YFP-tagged αS 3K (dox-inducible) were induced for the indicated time points (all cells plated at the same time, but induced at different time points), then fixed (4% paraformaldehyde) and imaged. Only very few smaller inclusions (

    Article Snippet: Plasmids pcDNA4/αS , pcDNA4/αS-3K , pcDNA4/αS-KLK , pcDNA4/αS-wt::YFP, pcDNA4/αS-3K::YFP , pcDNA4/αS-KLK::YFP , pcDNA4/αS-EIV::YFP ( ) and pCAX/dsRed ( ) have been described. αS-KLKEGR was synthesized as a GeneArt String DNA fragment (GeneArt/Life Technologies) and inserted into pcDNA4/TO/myc-His A (pcDNA4) with the In-Fusion HD Cloning Kit (Clontech).

    Techniques: Sequencing, Transfection, Selection, Western Blot, Positive Control, Negative Control, Over Expression, Expressing

    Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with DNA encoding SthK-bP and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .

    Journal: Frontiers in Neuroscience

    Article Title: Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition

    doi: 10.3389/fnins.2018.00643

    Figure Lengend Snippet: Characterization of a light-gated potassium channel in hippocampal neurons. (A) Maximum intensity projection of confocal images of hippocampal neurons 3 days after electroporation with DNA encoding SthK-bP and mKate2 (left: excitation 559 nm; right: excitation 488 nm). Lower images are close up of the regions indicated by dark boxes. (B) Single plane of the YFP signal of the region indicated in (A) (dark yellow box). (C) Left: Sample photocurrents evoked by a 50 ms light pulse (470 nm) of different intensity in a hippocampal neuron expressing mKate2 and SthK-bP. The holding potential was −70 mV. Right: Light intensity-response relationship fitted with a quadratic equation. Photocurrents are normalized to the maximum current recorded in each neuron. n = 5. (D) Left: Sample traces of photocurrents recorded from a SthK-bP expressing neuron when stimulated with 0.1 mW/mm 2 , 470 nm light (50 ms) while holding the cell at membrane potentials from −70 mV down to −115 mV. Baselines are aligned. Right: Current-voltage plot. A non-linear fit was applied to determine the K + equilibrium potential (−100.5 mV). (E) Photocurrent amplitude recorded from neurons expressing mKate2 and SthK-bP when stimulated with 1 mW mm −2 470 nm light. Shown are individual data points, median and 25–75% interquartile range, n = 5; median peak current SthK-bP 1.37 nA. (F) Kinetics of SthK-bP; data obtained from traces when the stimulation intensity was 1 mW mm −2 (470 nm, 50 ms); n = 5. (G) Whole-cell responses to current injections from −400 to 700 pA in SthK-bP expressing hippocampal neuron. (H) Action potentials generated by repeated somatic current injection (1,000 pA, 600 ms, ISI 5 s) were blocked for 3 min by 3 470 nm light flashes at 40 second intervals (50 ms at 1 mW mm −2 ); same neuron as in (G) .

    Article Snippet: The SthK channel DNA sequence was synthesized by GeneArt Strings DNA Fragments (Life technologies, Thermo Fisher Scientific) according to the published amino acid sequence (Brams et al., ; Kesters et al., ) with codon usage optimized for Mus musculus .

    Techniques: Electroporation, Mass Spectrometry, Expressing, Generated, Injection