fpg  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    New England Biolabs fpg
    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug <t>Fpg</t> or 1.2 µg/plug Nth for 24 h at <t>20°C</t> before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.
    Fpg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fpg/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fpg - by Bioz Stars, 2022-05
    95/100 stars

    Images

    1) Product Images from "Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair"

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr463

    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.
    Figure Legend Snippet: Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Techniques Used: Incubation, Irradiation

    2) Product Images from "Mitochondrial Functions Are Compromised in CD4 T Cells From ART-Controlled PLHIV"

    Article Title: Mitochondrial Functions Are Compromised in CD4 T Cells From ART-Controlled PLHIV

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.658420

    CD4 T cell mitochondrial functions in cART-controlled PLHIV and HS. (A) Frequency of MO + cells within CD4 + T cells from HIV-INRs, HIV-IRs, and HS. (B) Pearson’s correlation between the frequencies of MO + cells and CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (C) Spearman’s correlation between the frequencies of MO + cells in cycling CD4 + cells and cycling CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (D) Genomic DNA were purified from stimulated CD4 T cells, followed by qPCR to determine mtDNA relative to nuDNA (normalized to HS). (E) Genomic DNA were purified from stimulated CD4 T cells, treated with Fpg, followed by amplification of mtDNA by qPCR. (F, G) Representative OCR and summary data for non-mitochondrial, basal respiration, maximal respiration, spare capacity, proton leak, and ATP production in stimulated CD4 T cells from HIV-INRs, HIV-IRs, and HS. (H) ATP production was measured by a CellTiter-Glo luminescent assay in stimulated CD4 T cells from PLHIV and HS.
    Figure Legend Snippet: CD4 T cell mitochondrial functions in cART-controlled PLHIV and HS. (A) Frequency of MO + cells within CD4 + T cells from HIV-INRs, HIV-IRs, and HS. (B) Pearson’s correlation between the frequencies of MO + cells and CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (C) Spearman’s correlation between the frequencies of MO + cells in cycling CD4 + cells and cycling CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (D) Genomic DNA were purified from stimulated CD4 T cells, followed by qPCR to determine mtDNA relative to nuDNA (normalized to HS). (E) Genomic DNA were purified from stimulated CD4 T cells, treated with Fpg, followed by amplification of mtDNA by qPCR. (F, G) Representative OCR and summary data for non-mitochondrial, basal respiration, maximal respiration, spare capacity, proton leak, and ATP production in stimulated CD4 T cells from HIV-INRs, HIV-IRs, and HS. (H) ATP production was measured by a CellTiter-Glo luminescent assay in stimulated CD4 T cells from PLHIV and HS.

    Techniques Used: Purification, Real-time Polymerase Chain Reaction, Amplification, Luminescence Assay

    3) Product Images from "Rhus coriaria L. Fruit Extract Prevents UV-A-Induced Genotoxicity and Oxidative Injury in Human Microvascular Endothelial Cells"

    Article Title: Rhus coriaria L. Fruit Extract Prevents UV-A-Induced Genotoxicity and Oxidative Injury in Human Microvascular Endothelial Cells

    Journal: Antioxidants

    doi: 10.3390/antiox9040292

    Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using ( a ) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by ( b ) ENDO III for oxidized pyrimidines or ( c ) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm 2 UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p
    Figure Legend Snippet: Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using ( a ) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by ( b ) ENDO III for oxidized pyrimidines or ( c ) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm 2 UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p

    Techniques Used: Modification, Single Cell Gel Electrophoresis

    4) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).
    Figure Legend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation

    5) Product Images from "8-oxoguanine DNA glycosylase (OGG1) deficiency elicits coordinated changes in lipid and mitochondrial metabolism in muscle"

    Article Title: 8-oxoguanine DNA glycosylase (OGG1) deficiency elicits coordinated changes in lipid and mitochondrial metabolism in muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181687

    DNA damage estimations: mtDNA long amplicon PCR and total 8-oxoG content. A) Amplification of a long fragment of mtDNA was performed before and after treatment with FPG, a bacterial OGG1 functional analog. Data were normalized to amplification of a short mtDNA fragment. B) Total DNA was isolated by Miniprep and analyzed by ELISA for 8-oxoG content. Data are representative of 6 animals per genotype run in technical duplicates and expressed as mean ± SEM; , p
    Figure Legend Snippet: DNA damage estimations: mtDNA long amplicon PCR and total 8-oxoG content. A) Amplification of a long fragment of mtDNA was performed before and after treatment with FPG, a bacterial OGG1 functional analog. Data were normalized to amplification of a short mtDNA fragment. B) Total DNA was isolated by Miniprep and analyzed by ELISA for 8-oxoG content. Data are representative of 6 animals per genotype run in technical duplicates and expressed as mean ± SEM; , p

    Techniques Used: Amplification, Polymerase Chain Reaction, Functional Assay, Isolation, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Duplex-Repair enables highly accurate sequencing, despite DNA damage"

    Article Title: Duplex-Repair enables highly accurate sequencing, despite DNA damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab855

    Characterization of Duplex-Repair using capillary electrophoresis. ( A ) overview of Duplex-Repair vs. conventional ER/AT methods. ( B ) Schematic of the major products of various synthetic duplexes subjected to each step of Duplex-Repair and conventional ER/AT as determined by capillary electrophoresis (raw traces are in Fig S2). The non-fluorophore-tagged ends of the synthetic molecules are depicted, and fragment sizes are drawn to scale. Duplexes demarcated by asterisks (*) do not contain fluorophores and were not directly observed by capillary electrophoresis; however, their presence is predicted due to the characterized activities of UDG and FPG. Regions of strand resynthesis are illustrated in light blue.
    Figure Legend Snippet: Characterization of Duplex-Repair using capillary electrophoresis. ( A ) overview of Duplex-Repair vs. conventional ER/AT methods. ( B ) Schematic of the major products of various synthetic duplexes subjected to each step of Duplex-Repair and conventional ER/AT as determined by capillary electrophoresis (raw traces are in Fig S2). The non-fluorophore-tagged ends of the synthetic molecules are depicted, and fragment sizes are drawn to scale. Duplexes demarcated by asterisks (*) do not contain fluorophores and were not directly observed by capillary electrophoresis; however, their presence is predicted due to the characterized activities of UDG and FPG. Regions of strand resynthesis are illustrated in light blue.

    Techniques Used: Electrophoresis

    7) Product Images from "Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine"

    Article Title: Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw473

    Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.
    Figure Legend Snippet: Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Inhibition, Ligation

    8) Product Images from "The ubiquitin ligase RFWD3 is required for translesion DNA synthesis"

    Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2020.11.029

    RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).
    Figure Legend Snippet: RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).

    Techniques Used: Incubation, DNA Synthesis

    9) Product Images from "The ubiquitin ligase RFWD3 is required for translesion DNA synthesis"

    Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2020.11.029

    RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).
    Figure Legend Snippet: RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see Discussion ).

    Techniques Used: Incubation, DNA Synthesis

    10) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    11) Product Images from "Duplex-Repair enables highly accurate sequencing, despite DNA damage"

    Article Title: Duplex-Repair enables highly accurate sequencing, despite DNA damage

    Journal: bioRxiv

    doi: 10.1101/2021.05.21.445162

    Characterization of Duplex-Repair using capillary electrophoresis. (A) overview of Duplex-Repair vs. conventional ER/AT methods (B) Schematic of the major products of various synthetic duplexes subjected to each step of Duplex-Repair and conventional ER/AT as determined by capillary electrophoresis (Raw traces are in Fig S2). The non-fluorophore-tagged ends of the synthetic molecules are depicted, and fragment sizes are drawn to scale. Duplexes demarcated by asterisks (*) do not contain fluorophores and were not directly observed by capillary electrophoresis; however, their presence is predicted due to the characterized activities of UDG and FPG. Regions of strand resynthesis are illustrated in light blue.
    Figure Legend Snippet: Characterization of Duplex-Repair using capillary electrophoresis. (A) overview of Duplex-Repair vs. conventional ER/AT methods (B) Schematic of the major products of various synthetic duplexes subjected to each step of Duplex-Repair and conventional ER/AT as determined by capillary electrophoresis (Raw traces are in Fig S2). The non-fluorophore-tagged ends of the synthetic molecules are depicted, and fragment sizes are drawn to scale. Duplexes demarcated by asterisks (*) do not contain fluorophores and were not directly observed by capillary electrophoresis; however, their presence is predicted due to the characterized activities of UDG and FPG. Regions of strand resynthesis are illustrated in light blue.

    Techniques Used: Electrophoresis

    12) Product Images from "Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins"

    Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28642-9

    DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p
    Figure Legend Snippet: DDB2 facilitates 8-oxoG repair and is rapidly recruited to sites of 8-oxoG within telomeric DNA. a , b Immunofluorescence and quantification of 8-oxoG in cells transfected with control, DDB2 or OGG1 siRNA. c Schematic of the repair enzyme-based assay for 8-oxoG quantification in DNA. Genomic DNA containing 8-oxoG is treated with FPG to convert 8-oxoG to one nucleotide gaps. Treating with S1 nuclease converts the gaps to double stranded breaks (DSBs). The cleaved DNA is subjected to pulse field gel electrophoresis (PFGE) to track repair, as damaged DNA migrates faster than repaired DNA. d Quantification of 8-oxoG repair in U2OS cells transfected with control or DDB2 siRNA and treated with KBrO3. e Clonogenic cell survival curves in U2OS WT and DDB2 knockout (KO) cells treated with a range of concentrations of KBrO3. f Schematic of dye plus light treatment. Cells stably expressing FAP-TRF1 were treated with dye (100 nM, 15 min) plus light (660 nm, 10 min) to introduce 8-oxoG lesions at telomeres. g (left) Recruitment of DDB2-mCherry to 8-oxoG sites at telomeres in untreated, dye alone, light alone, and dye plus light treated cells. (right) Percentage telomeres colocalized with DDB2-mCherry. h Proximity ligation assay (PLA) for DDB2-mCherry and TRF1 in untreated cells and cells treated with dye (100 nM, 15 min) plus light (660 nm, 10 min). Data ( a , b , d , g , h ) represent mean ± SEM from two to three independent experiments. “ n ” represents the number of cells scored for each condition. Data ( e ) shows one representative experiment (performed in triplicate) from three independent experiments, mean ± SD. One-way ANOVA (Sidak multiple comparison test) ( b , g ), Student’s two-tailed Student’s t -test ( h ) and two-way ANOVA (Sidak multiple comparison test) ( d , e ) were performed for statistical analysis: * p

    Techniques Used: Immunofluorescence, Transfection, Enzymatic Assay, Nucleic Acid Electrophoresis, Knock-Out, Stable Transfection, Expressing, Introduce, Proximity Ligation Assay, Two Tailed Test

    13) Product Images from "HYPOXIA-INDUCED OXIDATIVE BASE MODIFICATIONS IN THE VEGF HYPOXIC RESPONSE ELEMENT ARE ASSOCIATED WITH TRANSCRIPTIONALLY ACTIVE NUCLEOSOMES"

    Article Title: HYPOXIA-INDUCED OXIDATIVE BASE MODIFICATIONS IN THE VEGF HYPOXIC RESPONSE ELEMENT ARE ASSOCIATED WITH TRANSCRIPTIONALLY ACTIVE NUCLEOSOMES

    Journal:

    doi: 10.1016/j.freeradbiomed.2008.09.038

    (A) PCR of treated and untreated with Fpg DNA fragments isolated from monomer, dimer, and trimer nucleosomal repeates and multi-nucleosomal zone with primers specific for the HRE of the VEGF promoter and 28S rRNA. (B) Pooled data for +/−Fpg PCR
    Figure Legend Snippet: (A) PCR of treated and untreated with Fpg DNA fragments isolated from monomer, dimer, and trimer nucleosomal repeates and multi-nucleosomal zone with primers specific for the HRE of the VEGF promoter and 28S rRNA. (B) Pooled data for +/−Fpg PCR

    Techniques Used: Polymerase Chain Reaction, Isolation

    (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without myxothiazol treatment. (B) PCR analysis of treated and untreated with Fpg
    Figure Legend Snippet: (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without myxothiazol treatment. (B) PCR analysis of treated and untreated with Fpg

    Techniques Used: Southern Blot, Polymerase Chain Reaction

    (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 or 30 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without TSA treatment. (B) PCR analysis of treated and untreated with Fpg DNA
    Figure Legend Snippet: (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 or 30 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without TSA treatment. (B) PCR analysis of treated and untreated with Fpg DNA

    Techniques Used: Southern Blot, Polymerase Chain Reaction

    14) Product Images from "Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair †"

    Article Title: Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair †

    Journal: Biochemistry

    doi: 10.1021/bi101674c

    Nucleotide incision of peroxynitrite-damaged plasmid DNA by Fpg and Mtb UvrA, UvrB, and UvrC proteins. (A) Plasmid DNA (0.5 mg/mL) was incubated with increasing concentrations of peroxynitrite (PN) in buffer containing 150 mM potassium phosphate buffer (pH 7.2) and 25 mM sodium bicarbonate. The DNA (500 ng) was resolved on 1% agarose gels, and percent nicked products was plotted vs concentration of PN. The data show means ± SD from three independent experiments (lower panel). Error bars fall within the symbols. (B) Plasmid (25 nM) treated with PN (open triangles, 50 μM; open circles, 200 μM) and incubated with increasing amounts of Fpg. Means ± SD from three independent experiments (lower panel). (C) Plasmid (25 nM) treated with PN (5.0 × 10 −5 M) was incubated with 100 nM UvrA, increasing concentrations of UvrB (0, 50, 100, 200, or 300 nM), and 150 nM UvrC. The data show means ± SD from three independent experiments (lower panel).
    Figure Legend Snippet: Nucleotide incision of peroxynitrite-damaged plasmid DNA by Fpg and Mtb UvrA, UvrB, and UvrC proteins. (A) Plasmid DNA (0.5 mg/mL) was incubated with increasing concentrations of peroxynitrite (PN) in buffer containing 150 mM potassium phosphate buffer (pH 7.2) and 25 mM sodium bicarbonate. The DNA (500 ng) was resolved on 1% agarose gels, and percent nicked products was plotted vs concentration of PN. The data show means ± SD from three independent experiments (lower panel). Error bars fall within the symbols. (B) Plasmid (25 nM) treated with PN (open triangles, 50 μM; open circles, 200 μM) and incubated with increasing amounts of Fpg. Means ± SD from three independent experiments (lower panel). (C) Plasmid (25 nM) treated with PN (5.0 × 10 −5 M) was incubated with 100 nM UvrA, increasing concentrations of UvrB (0, 50, 100, 200, or 300 nM), and 150 nM UvrC. The data show means ± SD from three independent experiments (lower panel).

    Techniques Used: Plasmid Preparation, Incubation, Concentration Assay

    15) Product Images from "The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells"

    Article Title: The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2010.10.692

    Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.
    Figure Legend Snippet: Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.

    Techniques Used: Single Cell Gel Electrophoresis, Transfection, Negative Control, Positive Control

    16) Product Images from "The Non-Bulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in vitro"

    Article Title: The Non-Bulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in vitro

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.7b00295

    Verification of template integrity of unmodified (UM), Gh and S -Sp modified linear templates by enzymatic digestion with I-PpoI and Fpg proteins. (A) Representative schematic of enzymatic digests to verify template integrity. I-PpoI digestion is used for verification of complete ligation of the DNA duplex to the CMV promoter fragment. The Fpg assay results in excision of the S- Sp and Gh lesions, generating 67 and 123 nucleotide fragments. (B): Representative denaturing gel showing the products of digestion of the linear templates with I-PpoI. (C) Representative denaturing gel showing the products of digestion first with I-PpoI, followed by treatment with Fpg-protein. Lanes M: oligodeoxynucleotide size markers (5′-[ 32 P]phosphate-labeled 50 bp Ladder (New England Biolabs)).
    Figure Legend Snippet: Verification of template integrity of unmodified (UM), Gh and S -Sp modified linear templates by enzymatic digestion with I-PpoI and Fpg proteins. (A) Representative schematic of enzymatic digests to verify template integrity. I-PpoI digestion is used for verification of complete ligation of the DNA duplex to the CMV promoter fragment. The Fpg assay results in excision of the S- Sp and Gh lesions, generating 67 and 123 nucleotide fragments. (B): Representative denaturing gel showing the products of digestion of the linear templates with I-PpoI. (C) Representative denaturing gel showing the products of digestion first with I-PpoI, followed by treatment with Fpg-protein. Lanes M: oligodeoxynucleotide size markers (5′-[ 32 P]phosphate-labeled 50 bp Ladder (New England Biolabs)).

    Techniques Used: Modification, Ligation, Labeling

    17) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    18) Product Images from "Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein"

    Article Title: Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2021.608839

    Schiff base trapping analysis of Fpg protein. DNA substrate (1 pmol) with either X⋅C (left panel) or X⋅G (right panel) base pair, alone as a negative control (lanes 1 and 2), or together with Fpg (10 pmol; lanes 3 and 4), was incubated with 50 mM NaBH 4 in reaction buffer at 37°C for 1 h (final volume, 10 μL). U⋅G-DNA (30 nt; 1 pmol) incubated alone (lane 5), with Ung (10 pmol; lane 6), or with Ung and Fpg (10 pmol each; lane 7), was used as a negative and positive control for active Ung and Fpg, respectively, Ung converting U⋅G-DNA into AP-DNA to be trapped by Fpg. The trapped protein was separated from un-trapped protein by denaturing PAGE. The experiments were performed 10 (X⋅C) or 5 times (X⋅G), the result being the same.
    Figure Legend Snippet: Schiff base trapping analysis of Fpg protein. DNA substrate (1 pmol) with either X⋅C (left panel) or X⋅G (right panel) base pair, alone as a negative control (lanes 1 and 2), or together with Fpg (10 pmol; lanes 3 and 4), was incubated with 50 mM NaBH 4 in reaction buffer at 37°C for 1 h (final volume, 10 μL). U⋅G-DNA (30 nt; 1 pmol) incubated alone (lane 5), with Ung (10 pmol; lane 6), or with Ung and Fpg (10 pmol each; lane 7), was used as a negative and positive control for active Ung and Fpg, respectively, Ung converting U⋅G-DNA into AP-DNA to be trapped by Fpg. The trapped protein was separated from un-trapped protein by denaturing PAGE. The experiments were performed 10 (X⋅C) or 5 times (X⋅G), the result being the same.

    Techniques Used: Negative Control, Incubation, Positive Control, Polyacrylamide Gel Electrophoresis

    Escherichia coli Fpg protein incises at unmethylated and methylated cytosine when placed opposite C and T in DNA. (A) Schematic representation of DNA substrates. Fluorescently labeled DNA oligonucleotides (*, phosphodiester bonds protected by phosphorothioate) had a spectrum of different studied bases at one site, as indicated. The upper strand was primarily labeled, its variable base defined by X (indicated in red); the lower strand was occasionally labeled, its variable base defined by Y (indicated in blue). The color code is kept throughout all the subsequent figures and tables indicating which variables in which strand were tested in a particular experiment. A variable base in the labeled strand (the assessed one) is always written as the first in a base pair. See Supplementary Figure 1 for an outline of the assay. (B) Activity for C opposite all major bases. (C) Activity for methylated Cs opposite C. (D) Activity for methylated Cs opposite T. In panels (B–D) , DNA (upper strand labeled) X substrate [see panel (A) , 1 pmol] was incubated alone (lanes 1–4) or with Fpg (13 pmol; lanes 5–8) at 37°C in NEB1 buffer (10 mM Bis–Tris-propane-HCl, pH 7.0, 10 mM MgCl 2 ), 1 mM DTT, 0.1 mg/mL BSA for 1 h. U⋅G-DNA (30 nt; 1 pmol) was incubated without (lane 9) or with Ung (1.95 pmol; lane 10) followed by NaOH/heat treatment, and was used as a negative and positive control, respectively, for active Ung, and to convert U⋅G-DNA into AP-DNA to demonstrate active Fpg (i.e., AP lyase activity; lane 11). (E) Activity for C and m 5 C opposite C, when the lower strand with the Y variable was labeled. DNA substrate (1 pmol) was incubated alone (lanes 1 and 2) or with Fpg (lanes 3 and 4) using the controls (lanes 5–7) described above. (F) Activity for C, m 5 C, or oxo 8 G opposite C compared to the other homo-mismatches. (G) Activity for C opposite C compared to Tg and dHT opposite A. In panels (F,G) , DNA (upper strand labeled) X substrate [see panel (A) , 1 pmol] was incubated alone (lanes 1–6 and 1–3, respectively) or with Fpg (lanes 7–12 and 4–6, respectively) using the controls (lanes 13–15 and 7–9, respectively) described above. (H) The percent of the labeled strand incised at unmethylated and methylated C opposite C. (I) The percent of the labeled strand incised at unmethylated C opposite T, A, or G compared to methylated C opposite T. (J) The percent of the labeled strand incised at most homo-mismatches compared to oxidized bases in DNA. These column graphs show the average values (±SD) obtained from 4 to 10 independent experiments as presented in panels (B–G) , the first base in each pair being the one assayed.
    Figure Legend Snippet: Escherichia coli Fpg protein incises at unmethylated and methylated cytosine when placed opposite C and T in DNA. (A) Schematic representation of DNA substrates. Fluorescently labeled DNA oligonucleotides (*, phosphodiester bonds protected by phosphorothioate) had a spectrum of different studied bases at one site, as indicated. The upper strand was primarily labeled, its variable base defined by X (indicated in red); the lower strand was occasionally labeled, its variable base defined by Y (indicated in blue). The color code is kept throughout all the subsequent figures and tables indicating which variables in which strand were tested in a particular experiment. A variable base in the labeled strand (the assessed one) is always written as the first in a base pair. See Supplementary Figure 1 for an outline of the assay. (B) Activity for C opposite all major bases. (C) Activity for methylated Cs opposite C. (D) Activity for methylated Cs opposite T. In panels (B–D) , DNA (upper strand labeled) X substrate [see panel (A) , 1 pmol] was incubated alone (lanes 1–4) or with Fpg (13 pmol; lanes 5–8) at 37°C in NEB1 buffer (10 mM Bis–Tris-propane-HCl, pH 7.0, 10 mM MgCl 2 ), 1 mM DTT, 0.1 mg/mL BSA for 1 h. U⋅G-DNA (30 nt; 1 pmol) was incubated without (lane 9) or with Ung (1.95 pmol; lane 10) followed by NaOH/heat treatment, and was used as a negative and positive control, respectively, for active Ung, and to convert U⋅G-DNA into AP-DNA to demonstrate active Fpg (i.e., AP lyase activity; lane 11). (E) Activity for C and m 5 C opposite C, when the lower strand with the Y variable was labeled. DNA substrate (1 pmol) was incubated alone (lanes 1 and 2) or with Fpg (lanes 3 and 4) using the controls (lanes 5–7) described above. (F) Activity for C, m 5 C, or oxo 8 G opposite C compared to the other homo-mismatches. (G) Activity for C opposite C compared to Tg and dHT opposite A. In panels (F,G) , DNA (upper strand labeled) X substrate [see panel (A) , 1 pmol] was incubated alone (lanes 1–6 and 1–3, respectively) or with Fpg (lanes 7–12 and 4–6, respectively) using the controls (lanes 13–15 and 7–9, respectively) described above. (H) The percent of the labeled strand incised at unmethylated and methylated C opposite C. (I) The percent of the labeled strand incised at unmethylated C opposite T, A, or G compared to methylated C opposite T. (J) The percent of the labeled strand incised at most homo-mismatches compared to oxidized bases in DNA. These column graphs show the average values (±SD) obtained from 4 to 10 independent experiments as presented in panels (B–G) , the first base in each pair being the one assayed.

    Techniques Used: Methylation, Labeling, Activity Assay, Incubation, Positive Control

    Definition and processing of the 3′-end after Fpg-mediated incision of m N 4,5 C⋅C-DNA. DNA substrate ( Figure 1A ; 1 pmol) was incubated without (lane 1) or with Fpg (13 pmol; lanes 2–4) at 37°C for 30 min, followed by no addition (lanes 1 and 2), addition of 0.083 pmol endonuclease IV (Nfo; lane 3) or addition of 0.29 pmol T4 polynucleotide kinase (PseT; lane 4), and incubation for an additional 30 min (final volume, 10 μL). Incised DNA was separated from un-incised DNA by denaturing PAGE ( Supplementary Figure 1 ) at 500 V for 4 h. Abbreviation: 3′-P, 3′-phosphate. The 5′-labeled strand is indicated by 5′ in magenta.
    Figure Legend Snippet: Definition and processing of the 3′-end after Fpg-mediated incision of m N 4,5 C⋅C-DNA. DNA substrate ( Figure 1A ; 1 pmol) was incubated without (lane 1) or with Fpg (13 pmol; lanes 2–4) at 37°C for 30 min, followed by no addition (lanes 1 and 2), addition of 0.083 pmol endonuclease IV (Nfo; lane 3) or addition of 0.29 pmol T4 polynucleotide kinase (PseT; lane 4), and incubation for an additional 30 min (final volume, 10 μL). Incised DNA was separated from un-incised DNA by denaturing PAGE ( Supplementary Figure 1 ) at 500 V for 4 h. Abbreviation: 3′-P, 3′-phosphate. The 5′-labeled strand is indicated by 5′ in magenta.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Labeling

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs fpg
    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug <t>Fpg</t> or 1.2 µg/plug Nth for 24 h at <t>20°C</t> before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.
    Fpg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fpg/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fpg - by Bioz Stars, 2022-05
    95/100 stars
      Buy from Supplier

    Image Search Results


    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Journal: Nucleic Acids Research

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    doi: 10.1093/nar/gkr463

    Figure Lengend Snippet: Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Article Snippet: Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ).

    Techniques: Incubation, Irradiation

    Percentage of bacterial DNA recognized by Fpg enzyme in model bacterial strains after ciprofloxacin, bleomycin, and cloxacillin treatment. The compounds were statistically significant at p

    Journal: Materials

    Article Title: Promiscuous Lipase-Catalyzed Markovnikov Addition of H-Phosphites to Vinyl Esters for the Synthesis of Cytotoxic α-Acyloxy Phosphonate Derivatives

    doi: 10.3390/ma15051975

    Figure Lengend Snippet: Percentage of bacterial DNA recognized by Fpg enzyme in model bacterial strains after ciprofloxacin, bleomycin, and cloxacillin treatment. The compounds were statistically significant at p

    Article Snippet: Dysfunction of bacterial membranes containing different lengths of LPS in model bacterial strains is an ideal model to assess the effectiveness of these compounds in relation to the antibiotics used by a specific enzyme Fpg of modified bacterial DNA (Labjot, New England Biolabs, Ipswich, MA, USA).

    Techniques:

    Percentage of plasmid DNA recognized by Fpg enzyme ( y -axis) with model bacterial, K12, and R2–R4 strains ( x -axis). All analyzed compounds numbered were statistically significant at

    Journal: Materials

    Article Title: Promiscuous Lipase-Catalyzed Markovnikov Addition of H-Phosphites to Vinyl Esters for the Synthesis of Cytotoxic α-Acyloxy Phosphonate Derivatives

    doi: 10.3390/ma15051975

    Figure Lengend Snippet: Percentage of plasmid DNA recognized by Fpg enzyme ( y -axis) with model bacterial, K12, and R2–R4 strains ( x -axis). All analyzed compounds numbered were statistically significant at

    Article Snippet: Dysfunction of bacterial membranes containing different lengths of LPS in model bacterial strains is an ideal model to assess the effectiveness of these compounds in relation to the antibiotics used by a specific enzyme Fpg of modified bacterial DNA (Labjot, New England Biolabs, Ipswich, MA, USA).

    Techniques: Plasmid Preparation

    CD4 T cell mitochondrial functions in cART-controlled PLHIV and HS. (A) Frequency of MO + cells within CD4 + T cells from HIV-INRs, HIV-IRs, and HS. (B) Pearson’s correlation between the frequencies of MO + cells and CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (C) Spearman’s correlation between the frequencies of MO + cells in cycling CD4 + cells and cycling CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (D) Genomic DNA were purified from stimulated CD4 T cells, followed by qPCR to determine mtDNA relative to nuDNA (normalized to HS). (E) Genomic DNA were purified from stimulated CD4 T cells, treated with Fpg, followed by amplification of mtDNA by qPCR. (F, G) Representative OCR and summary data for non-mitochondrial, basal respiration, maximal respiration, spare capacity, proton leak, and ATP production in stimulated CD4 T cells from HIV-INRs, HIV-IRs, and HS. (H) ATP production was measured by a CellTiter-Glo luminescent assay in stimulated CD4 T cells from PLHIV and HS.

    Journal: Frontiers in Immunology

    Article Title: Mitochondrial Functions Are Compromised in CD4 T Cells From ART-Controlled PLHIV

    doi: 10.3389/fimmu.2021.658420

    Figure Lengend Snippet: CD4 T cell mitochondrial functions in cART-controlled PLHIV and HS. (A) Frequency of MO + cells within CD4 + T cells from HIV-INRs, HIV-IRs, and HS. (B) Pearson’s correlation between the frequencies of MO + cells and CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (C) Spearman’s correlation between the frequencies of MO + cells in cycling CD4 + cells and cycling CD4 + T cells in HIV-INRs, HIV-IRs, and HS. (D) Genomic DNA were purified from stimulated CD4 T cells, followed by qPCR to determine mtDNA relative to nuDNA (normalized to HS). (E) Genomic DNA were purified from stimulated CD4 T cells, treated with Fpg, followed by amplification of mtDNA by qPCR. (F, G) Representative OCR and summary data for non-mitochondrial, basal respiration, maximal respiration, spare capacity, proton leak, and ATP production in stimulated CD4 T cells from HIV-INRs, HIV-IRs, and HS. (H) ATP production was measured by a CellTiter-Glo luminescent assay in stimulated CD4 T cells from PLHIV and HS.

    Article Snippet: For 8-oxoG quantification, 100 ng of DNA were treated with 10 units of Formamidopyrimidine glycosylase (Fpg, Cat# M0240L; New England Biolabs; Ipswich, MA) at 37°C for 1 h. Following digestion, 50 ng of template DNA were used for PCR.

    Techniques: Purification, Real-time Polymerase Chain Reaction, Amplification, Luminescence Assay

    Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using ( a ) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by ( b ) ENDO III for oxidized pyrimidines or ( c ) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm 2 UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p

    Journal: Antioxidants

    Article Title: Rhus coriaria L. Fruit Extract Prevents UV-A-Induced Genotoxicity and Oxidative Injury in Human Microvascular Endothelial Cells

    doi: 10.3390/antiox9040292

    Figure Lengend Snippet: Characterization of genotoxic damage induced by UV-A, and mERC extract’s role. Modified Comet assay measured direct DNA damage using ( a ) T4 PDG enzyme recognizing CPDs; indirect i.e., oxidative damage was identified by ( b ) ENDO III for oxidized pyrimidines or ( c ) FPG for oxidized purines. HMEC-1 cells were treated for 1 h with mERC (25 μg/mL, E25) and exposed to 20 J/cm 2 UV-A (T20). Results are expressed as mean of medians ± SEM, n = 3. Statistical analysis: One-Way ANOVA with Bonferroni’s post hoc analysis. ### p

    Article Snippet: The alkaline Comet assay was performed with some variations: the pH of the alkaline buffer was set at 12.1; after the lysis, slides were incubated for 45 min at 37 °C with 50 μL of the diluted enzymes: T4 PDG, ENDO III, FPG (New England BioLabs® Inc., 75-77 Knowl Piece, Wilbury Wai, Hitchin, UK).

    Techniques: Modification, Single Cell Gel Electrophoresis