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  • 93
    GE Healthcare nylon hybond n
    In vitro AUD RNA-binding activity. (A) E . coli expressed AUD (wildtype (WT) and P247A/V248A) were analysed by SDS-PAGE and Coomassie Brilliant blue staining. (B) Circular Dichroism analysis of purified AUD. (C) Schematic of the CHIKV genome showing the location of the various short RNA fragments used in subsequent filter binding analysis. (D-F) Filter binding analysis of the interaction between AUD and the indicated RNA fragments. Purified AUD at the indicated concentrations was incubated with radiolabelled RNA (1 nM) before application to a slot blot apparatus, filtering through nitrocellulose (protein-RNA complex) and <t>Hybond-N</t> (free RNA) membranes, and visualization by phosphoimaging. The negative control is wildtype AUD with an 80-mer aptamer raised against the foot-and-mouth disease virus 3D RNA-dependent RNA polymerase [ 18 ]. The percentage of RNA bound to the nitrocellulose membrane was quantified and plotted as a function of the AUD concentration. The data was fitted to a hyperbolic equation. Data are displayed as the means ± S.E. of three experimental replicates. (G) Endpoint (% of total RNA bound) and K d values derived from the graphs in (D-F).
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    GE Healthcare hybond n nylon filter
    RNA synthesis of CHIKV and SFV trans -replicases. BSR cells were co-transfected with plasmid encoding for wt or mutant CHIKV replicase and T7-Rluc-Tom encoding template for CHIKV replicase. Alternatively, cells were transfected with plasmids expressing SFV replicase and Tmed encoding for SFV specific template. Total RNA was isolated at 18 h p.t. Eight micrograms total RNA was fractionated and transferred to <t>Hybond-N+</t> nylon filter. Negative- and positive-strand RNAs were detected by hybridization with probes targeting the Rluc gene present in the template constructs. ( a ) Schematic presentation of T7-Rluc-Tom and Tmed plasmids encoding for replication competent template RNA of CHIKV and SFV. Plasmids have similar design except that number of codons from ORF1 of corresponding virus (designated as N) is 77 for T7-Rluc-Tom and 74 for Tmed. Other designations are the same as used for Fig. 5a . ( b ) Synthesis of negative-strand RNAs by SFV and CHIKV trans -replicases. ( c ) Synthesis of positive-strand full-length RNAs by SFV and CHIKV trans -replicases. Parts of the blots corresponding to template RNA are shown on panels b and c; for full-length images see Supplementary Information . The experiment was repeated trice with similar results.
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    89
    GE Healthcare nylon membrane hybond n
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    93
    Pall Corporation hybond n nylon membranes
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    Roche hybond n nylon membrane
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    92
    Thermo Fisher hybond n nylon membrane
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    GE Healthcare hybond n nylon paper
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    Nycomed hybond n nylon membrane
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    PerkinElmer hybond n nylon membranes
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    NEN Life Science hybond n nylon membrane
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    GE Healthcare hybond n blotting nylon membrane
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    GE Healthcare hybond n nylon membrane filter
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    GE Healthcare hybond n nylon hybridization membranes
    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to <t>Hybond-N</t> + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.
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    Image Search Results


    In vitro AUD RNA-binding activity. (A) E . coli expressed AUD (wildtype (WT) and P247A/V248A) were analysed by SDS-PAGE and Coomassie Brilliant blue staining. (B) Circular Dichroism analysis of purified AUD. (C) Schematic of the CHIKV genome showing the location of the various short RNA fragments used in subsequent filter binding analysis. (D-F) Filter binding analysis of the interaction between AUD and the indicated RNA fragments. Purified AUD at the indicated concentrations was incubated with radiolabelled RNA (1 nM) before application to a slot blot apparatus, filtering through nitrocellulose (protein-RNA complex) and Hybond-N (free RNA) membranes, and visualization by phosphoimaging. The negative control is wildtype AUD with an 80-mer aptamer raised against the foot-and-mouth disease virus 3D RNA-dependent RNA polymerase [ 18 ]. The percentage of RNA bound to the nitrocellulose membrane was quantified and plotted as a function of the AUD concentration. The data was fitted to a hyperbolic equation. Data are displayed as the means ± S.E. of three experimental replicates. (G) Endpoint (% of total RNA bound) and K d values derived from the graphs in (D-F).

    Journal: PLoS Pathogens

    Article Title: Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

    doi: 10.1371/journal.ppat.1007239

    Figure Lengend Snippet: In vitro AUD RNA-binding activity. (A) E . coli expressed AUD (wildtype (WT) and P247A/V248A) were analysed by SDS-PAGE and Coomassie Brilliant blue staining. (B) Circular Dichroism analysis of purified AUD. (C) Schematic of the CHIKV genome showing the location of the various short RNA fragments used in subsequent filter binding analysis. (D-F) Filter binding analysis of the interaction between AUD and the indicated RNA fragments. Purified AUD at the indicated concentrations was incubated with radiolabelled RNA (1 nM) before application to a slot blot apparatus, filtering through nitrocellulose (protein-RNA complex) and Hybond-N (free RNA) membranes, and visualization by phosphoimaging. The negative control is wildtype AUD with an 80-mer aptamer raised against the foot-and-mouth disease virus 3D RNA-dependent RNA polymerase [ 18 ]. The percentage of RNA bound to the nitrocellulose membrane was quantified and plotted as a function of the AUD concentration. The data was fitted to a hyperbolic equation. Data are displayed as the means ± S.E. of three experimental replicates. (G) Endpoint (% of total RNA bound) and K d values derived from the graphs in (D-F).

    Article Snippet: The binding reaction was initiated by mixing 1 nM radio-labelled RNA and AUD proteins (0 to 1 μM) in a 200 μl final volume at 4°C for 30 min. Membranes were pre-soaked in binding buffer supplemented with 5% (v/v) glycerol and assembled from bottom to top as follows in a slot-blot apparatus (Bio-Rad): filter paper, Hybond-N nylon (Amersham Biosciences) to bind free RNA molecules, and nitrocellulose (Schleicher & Schuell) to trap soluble protein-RNA complexes.

    Techniques: In Vitro, RNA Binding Assay, Activity Assay, SDS Page, Staining, Purification, Binding Assay, Incubation, Dot Blot, Negative Control, Concentration Assay, Derivative Assay

    RNA synthesis of CHIKV and SFV trans -replicases. BSR cells were co-transfected with plasmid encoding for wt or mutant CHIKV replicase and T7-Rluc-Tom encoding template for CHIKV replicase. Alternatively, cells were transfected with plasmids expressing SFV replicase and Tmed encoding for SFV specific template. Total RNA was isolated at 18 h p.t. Eight micrograms total RNA was fractionated and transferred to Hybond-N+ nylon filter. Negative- and positive-strand RNAs were detected by hybridization with probes targeting the Rluc gene present in the template constructs. ( a ) Schematic presentation of T7-Rluc-Tom and Tmed plasmids encoding for replication competent template RNA of CHIKV and SFV. Plasmids have similar design except that number of codons from ORF1 of corresponding virus (designated as N) is 77 for T7-Rluc-Tom and 74 for Tmed. Other designations are the same as used for Fig. 5a . ( b ) Synthesis of negative-strand RNAs by SFV and CHIKV trans -replicases. ( c ) Synthesis of positive-strand full-length RNAs by SFV and CHIKV trans -replicases. Parts of the blots corresponding to template RNA are shown on panels b and c; for full-length images see Supplementary Information . The experiment was repeated trice with similar results.

    Journal: Scientific Reports

    Article Title: Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

    doi: 10.1038/srep37124

    Figure Lengend Snippet: RNA synthesis of CHIKV and SFV trans -replicases. BSR cells were co-transfected with plasmid encoding for wt or mutant CHIKV replicase and T7-Rluc-Tom encoding template for CHIKV replicase. Alternatively, cells were transfected with plasmids expressing SFV replicase and Tmed encoding for SFV specific template. Total RNA was isolated at 18 h p.t. Eight micrograms total RNA was fractionated and transferred to Hybond-N+ nylon filter. Negative- and positive-strand RNAs were detected by hybridization with probes targeting the Rluc gene present in the template constructs. ( a ) Schematic presentation of T7-Rluc-Tom and Tmed plasmids encoding for replication competent template RNA of CHIKV and SFV. Plasmids have similar design except that number of codons from ORF1 of corresponding virus (designated as N) is 77 for T7-Rluc-Tom and 74 for Tmed. Other designations are the same as used for Fig. 5a . ( b ) Synthesis of negative-strand RNAs by SFV and CHIKV trans -replicases. ( c ) Synthesis of positive-strand full-length RNAs by SFV and CHIKV trans -replicases. Parts of the blots corresponding to template RNA are shown on panels b and c; for full-length images see Supplementary Information . The experiment was repeated trice with similar results.

    Article Snippet: Eight micrograms total RNA was fractionated on a denaturing 1% agarose gel and transferred to Hybond-N+ nylon filter (GE Healthcare).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Expressing, Isolation, Hybridization, Construct

    Differential expression of apoferritin light chain (ALC), cytochrome c oxidase I (CCO), TI-227H, and expressed in low invasive sarcoma cells (ELISC-1) between the highly invasive cell line GRU-1A and the low invasive cell line GRU-1B, as shown by northern blot analysis. Aliquots of 15 μg of total RNA were separated on a 1% agarose formaldehyde gel, blotted on to Hybond N nylon filters, and probed with DNA probes generated by PCR from the corresponding clone. Equal loading was confirmed by hybridisation with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. Whereas ALC was preferentially expressed by GRU-1A, the expression of CCO, TI-227, and ELISC-1 was higher in GRU-1B.

    Journal: Molecular Pathology

    Article Title: Differentially expressed genes in association with in vitro invasiveness of human epithelioid sarcoma

    doi:

    Figure Lengend Snippet: Differential expression of apoferritin light chain (ALC), cytochrome c oxidase I (CCO), TI-227H, and expressed in low invasive sarcoma cells (ELISC-1) between the highly invasive cell line GRU-1A and the low invasive cell line GRU-1B, as shown by northern blot analysis. Aliquots of 15 μg of total RNA were separated on a 1% agarose formaldehyde gel, blotted on to Hybond N nylon filters, and probed with DNA probes generated by PCR from the corresponding clone. Equal loading was confirmed by hybridisation with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe. Whereas ALC was preferentially expressed by GRU-1A, the expression of CCO, TI-227, and ELISC-1 was higher in GRU-1B.

    Article Snippet: Aliquots of 15 μg of total RNA were separated on a 1% agarose formaldehyde gel and blotted on to Hybond N nylon filters (Amersham, Little Chalfont, Buckinghamshire, UK).

    Techniques: Expressing, Northern Blot, Generated, Polymerase Chain Reaction, Hybridization

    (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to Hybond-N + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.

    Journal: Infection and Immunity

    Article Title: High Levels of Inducible Nitric Oxide Synthase mRNA Are Associated with Increased Monocyte Counts in Blood and Have a Beneficial Role in Plasmodium falciparum Malaria

    doi:

    Figure Lengend Snippet: (a) Slot blot semiquantitative analysis. RNAs isolated from peripheral whole blood from patients with falciparum malaria were reverse transcribed and subjected to 45 PCR cycles. Eight microliters from each reaction mixture was slot blotted as described in Materials and Methods. cDNA probes obtained from the PCRs were hybridized to PCR products, which were applied to Hybond-N + membranes with a slot blot manifold. Slots 1 to 12 contained PCR products from patients with severe and complicated malaria, and slots 13 to 24 contained PCR products from patients with uncomplicated malaria. Samples of patients with severe and complicated falciparum malaria (slots 1 to 12) were variably positive, with weak to moderate intensity. iNOS levels were markedly higher in patients with uncomplicated malaria than in those with complicated malaria. (b) Ratios of iNOs gene expression to β-actin gene expression. Densitometric values were determined from the slot blot shown in panel a and are presented as iNOS mRNA levels (percentages of β-actin). Samples are shown in the same order as in panel a.

    Article Snippet: Samples were rapidly cooled on ice and then blotted directly onto a Nylon membrane (Hybond-N+ ) (Amersham, Braunschweig, Germany) presoaked in 20× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2 PO4 , and 1 mM EDTA [pH 7.7]) in a slot blot apparatus (Minifold II; Schleicher & Schuell, Dassel, Germany).

    Techniques: Dot Blot, Isolation, Polymerase Chain Reaction, Expressing

    Replication of MVMp and H-1PV in human transformed or tumor cell lines. HEK293 and HEK293T (A), NB324K (B) and Hela (C) cells were mock-treated or infected with MVMp or H-1PV at 5 PFUs/cell. At the indicated time post-infection (p.i.) cells were harvested and DNA was extracted according to a modified Hirt procedure (see Materials and Methods ). Samples were then digested with proteinase K and 2 µg of DNA from each sample was then subjected to eletrophoresis through a 0.8% agarose gel and further transferred by capillarity on a Hybond-N membrane. Expression of DNA intermediates was investigated using a mixture of radiolabeled DNA probes corresponding to the E co RI-E co RV and H ind III-H ind III fragments of the viral NS genes from MVMp and H-1PV, respectively. Assessment of the migration of MVMp isolated genomes (0.08 µg) was used in each blot as migration control of the different DNA intermediates; mRF, monomeric replicative form; dRF, dimmer replicative form; ssDNA, single-stranded genome. The blots shown are representative of 3 experiments which all gave similar results.

    Journal: PLoS ONE

    Article Title: TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

    doi: 10.1371/journal.pone.0055086

    Figure Lengend Snippet: Replication of MVMp and H-1PV in human transformed or tumor cell lines. HEK293 and HEK293T (A), NB324K (B) and Hela (C) cells were mock-treated or infected with MVMp or H-1PV at 5 PFUs/cell. At the indicated time post-infection (p.i.) cells were harvested and DNA was extracted according to a modified Hirt procedure (see Materials and Methods ). Samples were then digested with proteinase K and 2 µg of DNA from each sample was then subjected to eletrophoresis through a 0.8% agarose gel and further transferred by capillarity on a Hybond-N membrane. Expression of DNA intermediates was investigated using a mixture of radiolabeled DNA probes corresponding to the E co RI-E co RV and H ind III-H ind III fragments of the viral NS genes from MVMp and H-1PV, respectively. Assessment of the migration of MVMp isolated genomes (0.08 µg) was used in each blot as migration control of the different DNA intermediates; mRF, monomeric replicative form; dRF, dimmer replicative form; ssDNA, single-stranded genome. The blots shown are representative of 3 experiments which all gave similar results.

    Article Snippet: After denaturation, the DNA was immobilized onto a nylon Hybond N+ membrane (Amersham Biosciences).

    Techniques: Transformation Assay, Infection, Modification, Agarose Gel Electrophoresis, Expressing, Migration, Isolation

    Northern blot analyses of selected ESTs under ABA treatment. Total RNA was denatured, transferred to Hybond-N + nylon membranes, and hybridized with DIG-labeled probes. LOX, lipoxygenase; HPP, hydroxyphenyl pyruvate integral membrane protein; XTH, xyloglucan endotransglucosylase/hydrolase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase; C, untreated control; 6 h, 12 h and 24 h indicate hours of ABA treatment.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Northern blot analyses of selected ESTs under ABA treatment. Total RNA was denatured, transferred to Hybond-N + nylon membranes, and hybridized with DIG-labeled probes. LOX, lipoxygenase; HPP, hydroxyphenyl pyruvate integral membrane protein; XTH, xyloglucan endotransglucosylase/hydrolase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase; C, untreated control; 6 h, 12 h and 24 h indicate hours of ABA treatment.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Northern Blot, Labeling

    Expression analyses of selected ESTs under osmotic stress. (A) Northern blot analysis. Total RNA was denatured, transferred to Hybond-N + nylon membranes and hybridized with DIG-labeled probes. (B) Expression analysis by RT-PCR. LEA, late embryogenesis abundant family protein; PEAMT, phosphoethanolamine methyltransferase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; XTH, xyloglucan endotransglucosylase/hydrolase; DHN, dehydrin; LOX, lipoxygenase; FNR, ferredoxin-NADP(H) oxidoreductase. C, unstressed control; 1 d, 2 d, 3 d, 5 d and 6 d indicate days of osmotic stress.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Expression analyses of selected ESTs under osmotic stress. (A) Northern blot analysis. Total RNA was denatured, transferred to Hybond-N + nylon membranes and hybridized with DIG-labeled probes. (B) Expression analysis by RT-PCR. LEA, late embryogenesis abundant family protein; PEAMT, phosphoethanolamine methyltransferase; ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; XTH, xyloglucan endotransglucosylase/hydrolase; DHN, dehydrin; LOX, lipoxygenase; FNR, ferredoxin-NADP(H) oxidoreductase. C, unstressed control; 1 d, 2 d, 3 d, 5 d and 6 d indicate days of osmotic stress.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Expressing, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction

    Confirmation of differential expression of clones from the osmotic stress SSH cDNA library. cDNAs from the forward subtraction were randomly picked and used to prepare two identical Hybond-N + nylon membranes. Clones were hybridized with (A) forward- or (B) reverse-subtracted probes.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Confirmation of differential expression of clones from the osmotic stress SSH cDNA library. cDNAs from the forward subtraction were randomly picked and used to prepare two identical Hybond-N + nylon membranes. Clones were hybridized with (A) forward- or (B) reverse-subtracted probes.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Expressing, Clone Assay, cDNA Library Assay

    Estimation of the copy number of genes for (A) ARF, (B) CIP and (C) PEAMT by Southern blot analysis. DNA was digested, electrophoresed, denatured, neutralized, transferred to Hybond-N + nylon membrane and hybridized with DIG-labeled probes. ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase.

    Journal: Breeding Science

    Article Title: Identification of osmotic stress-responsive genes from Leymus mollis, a wild relative of wheat (Triticum aestivum L.)

    doi: 10.1270/jsbbs.62.78

    Figure Lengend Snippet: Estimation of the copy number of genes for (A) ARF, (B) CIP and (C) PEAMT by Southern blot analysis. DNA was digested, electrophoresed, denatured, neutralized, transferred to Hybond-N + nylon membrane and hybridized with DIG-labeled probes. ARF, ETTIN-like auxin response factor; CIP, chloroplast inositol phosphate; PEAMT, phosphoethanolamine methyltransferase.

    Article Snippet: Southern and northern blot analyses and RT-PCR For northern blot analysis, total RNA (20 μg) from osmotic stressed or ABA-treated plants was denatured in formaldehyde gel and transferred to Hybond-N+ nylon membranes.

    Techniques: Southern Blot, Labeling