hcv pamp rna  (New England Biolabs)


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    Name:
    Antarctic Phosphatase
    Description:
    Antarctic Phosphatase 5 000 units
    Catalog Number:
    m0289l
    Price:
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    Size:
    5 000 units
    Category:
    Alkaline Phosphatases
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    New England Biolabs hcv pamp rna
    Antarctic Phosphatase
    Antarctic Phosphatase 5 000 units
    https://www.bioz.com/result/hcv pamp rna/product/New England Biolabs
    Average 85 stars, based on 623 article reviews
    Price from $9.99 to $1999.99
    hcv pamp rna - by Bioz Stars, 2020-07
    85/100 stars

    Images

    1) Product Images from "Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells"

    Article Title: Hepatitis C Virus Pathogen Associated Molecular Pattern (PAMP) Triggers Production of Lambda-Interferons by Human Plasmacytoid Dendritic Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003316

    RIG-I contributes to the recognition of the HCV PAMP RNA and is necessary for IFN gene production by GEN2.2. A) Gene expression graphs show that, after normalization to the reference gene GAPDH and Mock transfected condition, when compared to untreated HCV RNA, the phosphatases-treated RNA induced less interferon gene production. Data are representative graph from 3 independent experiments. Bars represent the mean and error bars are +/− SD. B) RIG-I was knocked down in pDC-GEN2.2 cells using siRNA and then stimulated with the pU/UC or X-region RNA. When compared to the Scrambled siRNA condition, the RIG-I knock-down condition (mean knock-down 13% by PCR) produced significantly less IFN mRNA. Combined data from 3 independent experiments. Bars represent the mean and error bars are +/− SEM. p values are the Mann-Whitney result for the scrambled siRNA condition compared to the RIG-I siRNA condition. * p
    Figure Legend Snippet: RIG-I contributes to the recognition of the HCV PAMP RNA and is necessary for IFN gene production by GEN2.2. A) Gene expression graphs show that, after normalization to the reference gene GAPDH and Mock transfected condition, when compared to untreated HCV RNA, the phosphatases-treated RNA induced less interferon gene production. Data are representative graph from 3 independent experiments. Bars represent the mean and error bars are +/− SD. B) RIG-I was knocked down in pDC-GEN2.2 cells using siRNA and then stimulated with the pU/UC or X-region RNA. When compared to the Scrambled siRNA condition, the RIG-I knock-down condition (mean knock-down 13% by PCR) produced significantly less IFN mRNA. Combined data from 3 independent experiments. Bars represent the mean and error bars are +/− SEM. p values are the Mann-Whitney result for the scrambled siRNA condition compared to the RIG-I siRNA condition. * p

    Techniques Used: Expressing, Transfection, Polymerase Chain Reaction, Produced, MANN-WHITNEY

    pDC-GEN2.2 cells sense HCV PAMP and produce Type I and Type III IFNs. A) Cartoon of the 3′ end of the HCV genome indicating the location of the poly U/UC (pU/UC, HCV PAMP) and the X-region in the 3′ UTR. Adapted from reference [20] . B) Kinetics of interferon gene upregulation in GEN2.2 cells following transfection with the pU/UC RNA. Fold increases for each gene at each condition are shown after normalization to reference gene GAPDH and are compared to transfection with the X-region RNA (dashed line). Levels of IFN expression at 2 (white bars), 4 (gray bars), 8 (hashed bars) or 24 (black bars) hours are shown for each gene. C) Kinetics of PRR genes and ISGs show upregulation by pU/UC stimulation. Kinetics are shown as indicated in A for the IFN genes. D–G) Secretion of Type I and III IFNs by pDC-GEN2.2 cells following PAMP-stimulation. Increased production of D) IFNα, E) IFNβ, F) IL-28A/IFNλ2 and G) IL-29/IFNλ1 as detected by ELISA from pDC-GEN2.2 cells that have been stimulated with HCV PAMP compared to the X-region RNA. H) Western Blot of pDC-GEN2.2 cell lysates for IL-28B/IFNλ3 after 24 hours of HCV PAMP stimulation. The antibody was specific for IL-28B/IFNλ3 as it recognized low levels of recombinant IL-28B/IFNλ3 (rIL-28B; 10 ng) but failed to recognize recombinant IL-28A/IFNλ2 (rIL-28A; 5 µg). I) pU/UC-stimulation increases PRR signaling proteins in accordance to gene expression data. Western blots of listed PRR proteins after 8 or 24 hours stimulation with HCV PAMP RNA. B–C) Combined data from 5 independent experiments. D–I) Combined data from 3 independent experiments except for the gene expression graphs which are 5 independent experiments. H–I) Representative blots from 3 independent experiments. Densitometry shows relative density of each band after normalization to the reference protein. For gene expression graphs, p values are the Wilcoxon signed rank result for each gene and time point compared to the X-region stimulation from the same gene and time point. For ELISA and Densitometry graphs, p values are the Mann-Whitney result for the pU/UC condition compared to the X-region condition. * p
    Figure Legend Snippet: pDC-GEN2.2 cells sense HCV PAMP and produce Type I and Type III IFNs. A) Cartoon of the 3′ end of the HCV genome indicating the location of the poly U/UC (pU/UC, HCV PAMP) and the X-region in the 3′ UTR. Adapted from reference [20] . B) Kinetics of interferon gene upregulation in GEN2.2 cells following transfection with the pU/UC RNA. Fold increases for each gene at each condition are shown after normalization to reference gene GAPDH and are compared to transfection with the X-region RNA (dashed line). Levels of IFN expression at 2 (white bars), 4 (gray bars), 8 (hashed bars) or 24 (black bars) hours are shown for each gene. C) Kinetics of PRR genes and ISGs show upregulation by pU/UC stimulation. Kinetics are shown as indicated in A for the IFN genes. D–G) Secretion of Type I and III IFNs by pDC-GEN2.2 cells following PAMP-stimulation. Increased production of D) IFNα, E) IFNβ, F) IL-28A/IFNλ2 and G) IL-29/IFNλ1 as detected by ELISA from pDC-GEN2.2 cells that have been stimulated with HCV PAMP compared to the X-region RNA. H) Western Blot of pDC-GEN2.2 cell lysates for IL-28B/IFNλ3 after 24 hours of HCV PAMP stimulation. The antibody was specific for IL-28B/IFNλ3 as it recognized low levels of recombinant IL-28B/IFNλ3 (rIL-28B; 10 ng) but failed to recognize recombinant IL-28A/IFNλ2 (rIL-28A; 5 µg). I) pU/UC-stimulation increases PRR signaling proteins in accordance to gene expression data. Western blots of listed PRR proteins after 8 or 24 hours stimulation with HCV PAMP RNA. B–C) Combined data from 5 independent experiments. D–I) Combined data from 3 independent experiments except for the gene expression graphs which are 5 independent experiments. H–I) Representative blots from 3 independent experiments. Densitometry shows relative density of each band after normalization to the reference protein. For gene expression graphs, p values are the Wilcoxon signed rank result for each gene and time point compared to the X-region stimulation from the same gene and time point. For ELISA and Densitometry graphs, p values are the Mann-Whitney result for the pU/UC condition compared to the X-region condition. * p

    Techniques Used: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, MANN-WHITNEY

    Co-culture of JFH-1-infected Huh7.5.1 cells and pDC-GEN2.2 cells leads to the upregulation of IFN genes and viral control. A) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then resting pDC-GEN2.2 cells were added for 24 hours. mRNA from the CD45+ cells was isolated and examined for IFN gene expression. Gene fold increase is shown for each gene after normalization to the reference gene GAPDH and the uninfected condition. B) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then pDC-GEN2.2 cells that had been mock transfected, transfected with X-region RNA or pU/UC RNA for 8 hours were added for 4 days (5 days total infection). RNA was isolated and examined for JFH-1 copy number. Normalized HCV copy number is shown where the infection control condition HCV copy number is set to 1 and other conditions are expressed as normalized HCV copy number compared to infection control. Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control) p values represent the Mann-Whitney result of the comparison. * p
    Figure Legend Snippet: Co-culture of JFH-1-infected Huh7.5.1 cells and pDC-GEN2.2 cells leads to the upregulation of IFN genes and viral control. A) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then resting pDC-GEN2.2 cells were added for 24 hours. mRNA from the CD45+ cells was isolated and examined for IFN gene expression. Gene fold increase is shown for each gene after normalization to the reference gene GAPDH and the uninfected condition. B) Huh7.5.1 cells were infected for 24 hours with JFH-1 and then pDC-GEN2.2 cells that had been mock transfected, transfected with X-region RNA or pU/UC RNA for 8 hours were added for 4 days (5 days total infection). RNA was isolated and examined for JFH-1 copy number. Normalized HCV copy number is shown where the infection control condition HCV copy number is set to 1 and other conditions are expressed as normalized HCV copy number compared to infection control. Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control) p values represent the Mann-Whitney result of the comparison. * p

    Techniques Used: Co-Culture Assay, Infection, Isolation, Expressing, Transfection, MANN-WHITNEY

    Ex vivo pDCs upregulate Type I and III Interferon genes in response to the HCV PAMP. A) Gene expression changes in ex vivo pDCs after HCV PAMP RNA stimulation. Top: subjects with the CC IL28B/IFNλ3 genotype (2 subjects IL28A/IFNλ2, IL28B/IFNλ3, IL29/IFNλ1 and IFNβ1; 1 subject IFNα2; RIG-I SNPs GG and GA); middle: CT IL28B/IFNλ3 genotype (1 subject, RIG-I SNP AA); Bottom: TT IL28B/IFNλ3 genotype (1 subject RIG-I SNP GG). Top graph is combined data from 2 independent experiments while middle and bottom graphs are data from a single independent experiment each. B) Same gene expression data as in A graphed together. Compared to the non-CC genotypes, the CC subjects had significantly more IFN mRNA. p values are the Wilcoxon signed rank result for (A) each gene compared to the X-region stimulation (dashed line) from the same gene, and (B) each gene CC genotype compared to each gene non-CC genotype. * p
    Figure Legend Snippet: Ex vivo pDCs upregulate Type I and III Interferon genes in response to the HCV PAMP. A) Gene expression changes in ex vivo pDCs after HCV PAMP RNA stimulation. Top: subjects with the CC IL28B/IFNλ3 genotype (2 subjects IL28A/IFNλ2, IL28B/IFNλ3, IL29/IFNλ1 and IFNβ1; 1 subject IFNα2; RIG-I SNPs GG and GA); middle: CT IL28B/IFNλ3 genotype (1 subject, RIG-I SNP AA); Bottom: TT IL28B/IFNλ3 genotype (1 subject RIG-I SNP GG). Top graph is combined data from 2 independent experiments while middle and bottom graphs are data from a single independent experiment each. B) Same gene expression data as in A graphed together. Compared to the non-CC genotypes, the CC subjects had significantly more IFN mRNA. p values are the Wilcoxon signed rank result for (A) each gene compared to the X-region stimulation (dashed line) from the same gene, and (B) each gene CC genotype compared to each gene non-CC genotype. * p

    Techniques Used: Ex Vivo, Expressing

    Replicative control of HCV in JFH-1/Huh7.5.1 system with conditioned media (CM) from pU/UC-transfected pDC-GEN2.2 cells. Huh7.5.1 cells were infected for 24 hours prior to the addition of CM (A–D) or rIFNs (E) then 4 days later (5 days post-infection), cells were lysed and examined for HCV Copy number by qRT-PCR (see methods). A) Normalized JFH-1 copy number (see methods and below for calculation) after treatment with CM from Mock (negative; white bars)-, X-region (gray bars)- or pU/UC (dark gray bars)-stimulated pDC-GEN2.2 cells after 8 hours of RNA stimulation. B) Dose-dependent response of viral replication control. CM from the HCV PAMP-stimulated pDC-GEN2.2 cells was added to JFH-1 infected cells at the following dilutions: 1∶1 (Dark gray bars), 1∶10 (gray bars) or 1∶100 (white bars). C) Type I IFN dependence was determined using the Vaccinia protein B18R which blocks Type I IFN responses. D) Blocking IL-28B/IL-29 (IFNλ3/IFNλ1) with a blocking, cross-reactive antibody demonstrates dependence on Type III IFNs for a portion of the viral control. E) Recombinant Type III Interferons in the absence of CM at the same concentrations as found in the CM (IL-28A/IFNλ2: 1500 pg/mL; IL-28B/IFNλ3: 10 pg/mL; IL-29/IFNλ1: 500 pg/mL) were added to JFH-1 infected Huh7.5.1 cells. Normalized HCV Copy Number is shown where the infection control condition HCV copy number is set to 1 (except panel A where the Mock condition is set to 1) and other conditions are expressed as normalized HCV copy number compared to infection control (or compared to Mock in panel A). Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control). Combined data from 3 (A, C and E), 5 (B, D) independent experiments p values represent the Mann-Whitney result of the comparison. * p
    Figure Legend Snippet: Replicative control of HCV in JFH-1/Huh7.5.1 system with conditioned media (CM) from pU/UC-transfected pDC-GEN2.2 cells. Huh7.5.1 cells were infected for 24 hours prior to the addition of CM (A–D) or rIFNs (E) then 4 days later (5 days post-infection), cells were lysed and examined for HCV Copy number by qRT-PCR (see methods). A) Normalized JFH-1 copy number (see methods and below for calculation) after treatment with CM from Mock (negative; white bars)-, X-region (gray bars)- or pU/UC (dark gray bars)-stimulated pDC-GEN2.2 cells after 8 hours of RNA stimulation. B) Dose-dependent response of viral replication control. CM from the HCV PAMP-stimulated pDC-GEN2.2 cells was added to JFH-1 infected cells at the following dilutions: 1∶1 (Dark gray bars), 1∶10 (gray bars) or 1∶100 (white bars). C) Type I IFN dependence was determined using the Vaccinia protein B18R which blocks Type I IFN responses. D) Blocking IL-28B/IL-29 (IFNλ3/IFNλ1) with a blocking, cross-reactive antibody demonstrates dependence on Type III IFNs for a portion of the viral control. E) Recombinant Type III Interferons in the absence of CM at the same concentrations as found in the CM (IL-28A/IFNλ2: 1500 pg/mL; IL-28B/IFNλ3: 10 pg/mL; IL-29/IFNλ1: 500 pg/mL) were added to JFH-1 infected Huh7.5.1 cells. Normalized HCV Copy Number is shown where the infection control condition HCV copy number is set to 1 (except panel A where the Mock condition is set to 1) and other conditions are expressed as normalized HCV copy number compared to infection control (or compared to Mock in panel A). Normalized HCV Copy Number = (Absolute copy number for condition/absolute copy number for infection control). Combined data from 3 (A, C and E), 5 (B, D) independent experiments p values represent the Mann-Whitney result of the comparison. * p

    Techniques Used: Transfection, Infection, Quantitative RT-PCR, Blocking Assay, Recombinant, MANN-WHITNEY

    2) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).
    Figure Legend Snippet: Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).

    Techniques Used: Modification, Injection, Isolation, Expressing, Polymerase Chain Reaction, Amplification

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    3) Product Images from "Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases"

    Article Title: Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.612929

    RNA 3 ′ -terminal phosphate modifications with MthRnl. A , gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p ( column I ) or FAM-RNA17(OMe)p ( column II ) followed by phosphatase treatment with either AnP or PNK.
    Figure Legend Snippet: RNA 3 ′ -terminal phosphate modifications with MthRnl. A , gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p ( column I ) or FAM-RNA17(OMe)p ( column II ) followed by phosphatase treatment with either AnP or PNK.

    Techniques Used: Electrophoretic Mobility Shift Assay, Aqueous Normal-phase Chromatography

    4) Product Images from "Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α"

    Article Title: Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00067-16

    Destabilization of B-Myb by pVHL. (A) HEK293T cells expressing 3×HA–B-Myb with or without 3×FLAG-pVHL were exposed to cycloheximide (CHX [50 μg/ml]) for 1, 2, or 4 h. The lysates were subjected to Western blotting with
    Figure Legend Snippet: Destabilization of B-Myb by pVHL. (A) HEK293T cells expressing 3×HA–B-Myb with or without 3×FLAG-pVHL were exposed to cycloheximide (CHX [50 μg/ml]) for 1, 2, or 4 h. The lysates were subjected to Western blotting with

    Techniques Used: Expressing, Western Blot

    Regulation of B-Myb stability by phosphorylation of tyrosine 15. (A) Tyrosine phosphorylation of B-Myb under sparse culture conditions. 786-O cells (2.6 × 10 6 ) stably expressing 3×HA–B-Myb were cultured under confluent (in one
    Figure Legend Snippet: Regulation of B-Myb stability by phosphorylation of tyrosine 15. (A) Tyrosine phosphorylation of B-Myb under sparse culture conditions. 786-O cells (2.6 × 10 6 ) stably expressing 3×HA–B-Myb were cultured under confluent (in one

    Techniques Used: Stable Transfection, Expressing, Cell Culture

    Regulation of B-Myb by VEGF and PDGF. (A) VEGFR and PDGFR inhibitor prevents tyrosine phosphorylation of B-Myb. 786-O cells stably expressing 3×HA–B-Myb or control cells were sparsely cultured for 1 day, followed by RTK inhibitor treatment
    Figure Legend Snippet: Regulation of B-Myb by VEGF and PDGF. (A) VEGFR and PDGFR inhibitor prevents tyrosine phosphorylation of B-Myb. 786-O cells stably expressing 3×HA–B-Myb or control cells were sparsely cultured for 1 day, followed by RTK inhibitor treatment

    Techniques Used: Stable Transfection, Expressing, Cell Culture

    5) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).
    Figure Legend Snippet: Modification of the crucial steps of the DamID protocol. (A) Medaka zygotes were injected with mRNA coding for Dam-f-GFP or Dam-f-TF (Medaka Rx2). Embryos were maintained in ERM supplemented with an antibiotic solution and gDNA was isolated at stage 22. (B) Medaka embryos (stage 22) expressing Dam-f-GFP. (C) DamID LM-PCR at 25 cycles using the modifications presented in the main text generates only Dpn I-dependent amplification (see Materials and Methods, iDamIDseq protocol). (D) Flowchart comparing the standard DamID-seq protocol (based on Wu et al., 2016 ) with the iDamIDseq protocol (improvements are underlined).

    Techniques Used: Modification, Injection, Isolation, Expressing, Polymerase Chain Reaction, Amplification

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    6) Product Images from "Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract"

    Article Title: Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract

    Journal: Microbiology

    doi: 10.1099/mic.0.000515

    Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.
    Figure Legend Snippet: Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.

    Techniques Used: Western Blot, Hybridization, Marker

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A Non-invasive Radiomic Method Using 18F-FDG PET Predicts Isocitrate Dehydrogenase Genotype and Prognosis in Patients With Glioma
    Article Snippet: .. PCR products were treated with Exonuclease I and Antarctic Phosphatase (New England Biolabs, UK) and sequenced using a Genetic Analyzers 3500 (Thermo Fisher, US). .. 18 F-FDG PET Data Acquisition and Tumor Segmentation 18 F-FDG was produced in situ using an RDS-111 Cyclotron (CTI, US).

    Aqueous Normal-phase Chromatography:

    Article Title: Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases
    Article Snippet: .. Antarctic phosphatase (AnP), T4 PNK, and yeast 5′Deadenylase were from New England Biolabs. ..

    Subcloning:

    Article Title: Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H ▿
    Article Snippet: .. This was done by subcloning a BamHI/KpnI genomic fragment (coordinates 64765 to 66120) from the BamHI-N genomic clone ( ) into the BamHI/KpnI sites of pSP73, digesting it with AccI, blunting with Klenow fragment DNA polymerase, and dephosphorylating with Antarctic alkaline phosphatase (New England Biolabs). .. The phosphorylated oligonucleotide was then ligated in to terminate gL translation 2 amino acids before its predicted signal sequence cleavage site (gL- STOP).

    Modification:

    Article Title: METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation
    Article Snippet: .. Mass spectrometry analysis of RNA nucleoside m7G modification Nucleosides were prepared from enzyme-processed RNA by enzymatic digestion, using a cocktail of Benzonase (Merck), Phosphodiesterase 1 (Merck), and Antarctic Phosphatase (New England Biolabs) as described previously ( ). .. The reactions were filtered using an Amicon 30kDa MWCO spin-column (Merck) to remove protein and the filtrate was mixed with a 2x loading buffer containing 0.1% formic acid and an internal standard (13 C-labeled uridine generated from 645672-1MG Merck KGaA, previously treated with Antarctic Phosphatase).

    Mass Spectrometry:

    Article Title: METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation
    Article Snippet: .. Mass spectrometry analysis of RNA nucleoside m7G modification Nucleosides were prepared from enzyme-processed RNA by enzymatic digestion, using a cocktail of Benzonase (Merck), Phosphodiesterase 1 (Merck), and Antarctic Phosphatase (New England Biolabs) as described previously ( ). .. The reactions were filtered using an Amicon 30kDa MWCO spin-column (Merck) to remove protein and the filtrate was mixed with a 2x loading buffer containing 0.1% formic acid and an internal standard (13 C-labeled uridine generated from 645672-1MG Merck KGaA, previously treated with Antarctic Phosphatase).

    Sequencing:

    Article Title: Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract
    Article Snippet: .. A 17 438 bp sequence containing the novel lantibiotic cluster was restricted from the identified pJAZZ-OC clone with ClaI and PstI (NEB) and then ligated into vector pIL253 [MspI, PstI restricted and dephosphorylated (Antarctic Phosphatase, NEB)] using Fastlink DNA ligase (Epicentre) to create p nso. .. The construct was transformed into electrocompetent L. lactis MG1614 using a Gene Pulse Xcell (BioRad, [ ]).

    Plasmid Preparation:

    Article Title: Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract
    Article Snippet: .. A 17 438 bp sequence containing the novel lantibiotic cluster was restricted from the identified pJAZZ-OC clone with ClaI and PstI (NEB) and then ligated into vector pIL253 [MspI, PstI restricted and dephosphorylated (Antarctic Phosphatase, NEB)] using Fastlink DNA ligase (Epicentre) to create p nso. .. The construct was transformed into electrocompetent L. lactis MG1614 using a Gene Pulse Xcell (BioRad, [ ]).

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    New England Biolabs antarctic phosphatase anp
    RNA 3 ′ -terminal phosphate modifications with MthRnl. A , gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p ( column I ) or FAM-RNA17(OMe)p ( column II ) followed by phosphatase treatment with either <t>AnP</t> or <t>PNK.</t>
    Antarctic Phosphatase Anp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RNA 3 ′ -terminal phosphate modifications with MthRnl. A , gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p ( column I ) or FAM-RNA17(OMe)p ( column II ) followed by phosphatase treatment with either AnP or PNK.

    Journal: The Journal of Biological Chemistry

    Article Title: Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

    doi: 10.1074/jbc.M114.612929

    Figure Lengend Snippet: RNA 3 ′ -terminal phosphate modifications with MthRnl. A , gel shift analysis of archaeal RNA ligase (MthRnl) reaction products of either FAM-RNA17p ( column I ) or FAM-RNA17(OMe)p ( column II ) followed by phosphatase treatment with either AnP or PNK.

    Article Snippet: Antarctic phosphatase (AnP), T4 PNK, and yeast 5′Deadenylase were from New England Biolabs.

    Techniques: Electrophoretic Mobility Shift Assay, Aqueous Normal-phase Chromatography

    Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.

    Journal: Microbiology

    Article Title: Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract

    doi: 10.1099/mic.0.000515

    Figure Lengend Snippet: Western hybridization using the NsoA1 leader antibody to detect pre-peptide production in UKLc10. Comparison of L. lactis TCA-precipitated culture supernatant extracts (lanes 1–5) or cell extracts (lanes 6–11) from UKLc10 (lanes 1, 2, 4–8, 10 and 11) or MG1614 (lanes 3 and 9) containing plasmids p nsoA Δ A (lanes 1 and 11), p nsoA (lanes 2, 6 and 10), p nsoA Δ A , pTG nsoA3-nsoA4 (lanes 3 and 9), pIL253 (lanes 4 and 8) and p nsoA pTG nsoA3-nsoA4 (lanes 5 and 7). Samples were induced with nisin for 3 h, except for lane 6 (2 h). M, marker.

    Article Snippet: A 17 438 bp sequence containing the novel lantibiotic cluster was restricted from the identified pJAZZ-OC clone with ClaI and PstI (NEB) and then ligated into vector pIL253 [MspI, PstI restricted and dephosphorylated (Antarctic Phosphatase, NEB)] using Fastlink DNA ligase (Epicentre) to create p nso.

    Techniques: Western Blot, Hybridization, Marker