fpg  (New England Biolabs)


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  • 99
    Name:
    Fpg
    Description:
    Fpg 2 500 units
    Catalog Number:
    M0240L
    Price:
    296
    Size:
    2 500 units
    Category:
    Other Enzymes
    Score:
    85
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    Structured Review

    New England Biolabs fpg
    Fpg
    Fpg 2 500 units
    https://www.bioz.com/result/fpg/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fpg - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    Journal: Genes and Environment

    doi: 10.1186/s41021-018-0112-5

    Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows
    Figure Legend Snippet: Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows

    Techniques Used: Sequencing, Purification, Incubation, Agarose Gel Electrophoresis, Staining, Marker, Countercurrent Chromatography

    6) 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

    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 "HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy"

    Article Title: HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp375

    Characterization of the AP lyase activity. ( A ) Creation of AP substrate containing a single abasic site using uracil DNA glycosylase (UDG). ( B ) 15% denaturing SDS–PAGE. Four micrograms per lane of each protein was loaded onto the gel. ( C ) Determination of the chemical nature of DNA ends generated by HMGA2. Cleavage products obtained with the following E. coli proteins: formamidopyrimidine N -glycosylase (Fpg), endonucleases III (endo III) and IV (endo IV), as indicated, can be separated from substrates through denaturing polyacrylamide gel electrophoresis. The 31-mer incubated under the same conditions, but in the absence of protein is shown as control in lane 1. ( D ) Quantification of AP lyase activities. After electrophoresis and autoradiography, bands corresponding to substrate and products were quantified and plotted against the molar protein to DNA ratio.
    Figure Legend Snippet: Characterization of the AP lyase activity. ( A ) Creation of AP substrate containing a single abasic site using uracil DNA glycosylase (UDG). ( B ) 15% denaturing SDS–PAGE. Four micrograms per lane of each protein was loaded onto the gel. ( C ) Determination of the chemical nature of DNA ends generated by HMGA2. Cleavage products obtained with the following E. coli proteins: formamidopyrimidine N -glycosylase (Fpg), endonucleases III (endo III) and IV (endo IV), as indicated, can be separated from substrates through denaturing polyacrylamide gel electrophoresis. The 31-mer incubated under the same conditions, but in the absence of protein is shown as control in lane 1. ( D ) Quantification of AP lyase activities. After electrophoresis and autoradiography, bands corresponding to substrate and products were quantified and plotted against the molar protein to DNA ratio.

    Techniques Used: Activity Assay, SDS Page, Generated, Polyacrylamide Gel Electrophoresis, Incubation, Electrophoresis, Autoradiography

    Related Articles

    Amplification:

    Article Title: Perinuclear Mitochondrial Clustering Creates an Oxidant-Rich Nuclear Domain Required for Hypoxia-Induced Transcription
    Article Snippet: Hypoxia-induced oxidative base modifications in selected HRE sequences were detected with a previously described PCR-based technique wherein the bacterial DNA glycosylase Fpg (New England Biolabs) was applied to recognize and cleave 8-oxoguanine and related oxidized base products ( , ). .. Treatment of DNA with Fpg results in strand cleavage at sites of oxidized purines, thereby creating single-strand breaks that block PCR amplification.

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg. .. In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg.

    Positive Control:

    Article Title: Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats
    Article Snippet: DNA glycosylase reactions were performed by mixing purified protein as indicated, with 10–50 fmol DNA substrate in a total volume of 15 μL. .. The enzyme activities were assayed in a reaction buffer containing 70 mM 3-(N -morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 5% glycerol, and incubated at 37 °C for 1 h. Fpg Ec (New England Biolabs) was included as the positive control. .. Products of the reactions were separated by 20% denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by phosphorimaging.

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: As a negative control, cells were incubated in LHC9 medium alone, while as a positive control cells were exposed (5 min at 4 °C, protected from light) to 110 μM hydrogen peroxide (H2 O2 ). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Neutralization:

    Article Title:
    Article Snippet: The slides were then washed (three 10 min washes) with neutralization buffer (0.4 m Tris, pH 7.4), followed by a 10-min incubation with REC buffer (10 m m HEPES-KOH, 100 m m KCl, 10 m m EDTA, 0.1 mg/ml BSA, pH 7.4). .. Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: The slide was incubated in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton X-100) for at least 4 hours then washed with neutralization buffer (0.4 M Tris, pH 7.4), followed by a 10 minute incubation with REC buffer (10 mM HEPES-KOH, 100 mM KCl, 10 mM EDTA, 0.1 mg/ml BSA; pH 7.4). .. For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes.

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: After neutralization and dehydration, slides were air-dried before being stained with propidium iodide (PI). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Picogreen Assay:

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: Digested DNA samples were precipitated, dissolved in TE buffer, and precisely quantified on the Bio-Rad Versa Fluor fluorometer (Bio-Rad Laboratories, Hercules, CA) using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA). .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines.

    Construct:

    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
    Article Snippet: Purified DNA pellets were resuspended in buffer TE, pH 8.0 and yields were determined using NanoDrop spectrophotometry. .. 250 ng of each construct were digested with Formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs, Cat. #M0240S) using 1 μL of Fpg (8 units) in the presence of BSA, according to the manufacturer’s instructions, in 1X NEBuffer 1 for 1 hr at 37°C. .. Products were separated and visualized on 0.7% agarose TBE gels containing ethidium bromide.

    Real-time Polymerase Chain Reaction:

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: Quantitative real time PCR was employed to determine the intact DNA level of specific amplicons within both the human and mouse p62 promoter. .. In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg.

    Incubation:

    Article Title: An automated Fpg-based FADU method for the detection of oxidative DNA lesions and screening of antioxidants
    Article Snippet: Samples were then divided into aliquots of 4 µg and 100 µg of DNA for FADU and LC–MS analysis, respectively. .. Each sample was incubated with 8 U Fpg and NEB1 Buffer (New England Biolabs) for 30 min at 30 °C. .. Samples were diluted in 280 µl FADU suspension buffer (250 mM meso -inositol, 10 mM sodium phosphate, 1 mM MgCl, pH 7.4) and quadruplicates were transferred into a 96-well plate which was positioned into the FADU pipetting robot.

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Following T4pdg treatment, samples were loaded into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs. .. The fluorescence emission spectrum was measured before addition of probe to determine buffer fluorescence.

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: Basically the same procedure was applied for the measurement of DNA base damage. .. The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in . .. Briefly, after lysis, the slides were washed 3 × 5 min with the FPG buffer (40 mM Hepes (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL bovine serum albumin, pH 8) at 4 °C.

    Article Title: Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats
    Article Snippet: DNA glycosylase reactions were performed by mixing purified protein as indicated, with 10–50 fmol DNA substrate in a total volume of 15 μL. .. The enzyme activities were assayed in a reaction buffer containing 70 mM 3-(N -morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 5% glycerol, and incubated at 37 °C for 1 h. Fpg Ec (New England Biolabs) was included as the positive control. .. Products of the reactions were separated by 20% denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by phosphorimaging.

    Article Title:
    Article Snippet: The slides were then washed (three 10 min washes) with neutralization buffer (0.4 m Tris, pH 7.4), followed by a 10-min incubation with REC buffer (10 m m HEPES-KOH, 100 m m KCl, 10 m m EDTA, 0.1 mg/ml BSA, pH 7.4). .. Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA. .. Electrophoresis was then performed at 25 volts for 15 min, and the slides were dehydrated in 100% ethanol for 5 min, and then stained with ethidium bromide (10 ng/ml).

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: Primers designed for each amplicon are shown in . .. In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg. .. An aliquot of the reaction mixture was used for quantitative PCR assay.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: The slide was incubated in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton X-100) for at least 4 hours then washed with neutralization buffer (0.4 M Tris, pH 7.4), followed by a 10 minute incubation with REC buffer (10 mM HEPES-KOH, 100 mM KCl, 10 mM EDTA, 0.1 mg/ml BSA; pH 7.4). .. For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. Fpg treatment was used on samples exposed to hydrogen peroxide and related controls, and Aag treatment on MMS-exposed sets and related controls.

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1. .. Following T4pdg treatment, samples were loaded into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V).

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: As a negative control, cells were incubated in LHC9 medium alone, while as a positive control cells were exposed (5 min at 4 °C, protected from light) to 110 μM hydrogen peroxide (H2 O2 ). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C. .. Slides, which were prepared in duplicate for each experimental condition, were analyzed under a fluorescence microscope at 400 X magnification using the Komet 6.0 software (Andor Bioimaging, Nottingham, UK).

    Single Cell Gel Electrophoresis:

    Article Title: Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2
    Article Snippet: After lysis, FPG and buffer slides were washed three times with cold enzyme buffer (40 mM HEPES, 0.1 M KCL, 0.5 mM EDTA, 0.2 mg/mL BSA, pH 8) before being clamped into slide units (12-Gel Comet Assay Unit™ , Severn Biotech Limited). .. The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH).

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in . .. The slides were stained with DAPI (1 μg/mL) and analysed as described above.

    Article Title:
    Article Snippet: Paragraph title: Alkali Single-cell Gel Electrophoresis (Comet Assay) ... Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: Paragraph title: 2.4 Alkali single-cell gel electrophoresis (comet assay) ... For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes.

    Derivative Assay:

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg. .. All reactions were performed in a 25 μl mixture containing 1X SYBR Green master mix, 0.2 μM primer mix (forward and reverse), and template DNA for QPCR, respectively.

    Southern Blot:

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines.

    SYBR Green Assay:

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg. .. An aliquot of the reaction mixture was used for quantitative PCR assay.

    Generated:

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: The initial velocity was determined by measuring the rate of fluorescence increase at 460 nm over the first 10 minutes of the reaction, during which the fluorescence increase was linear. .. Michaelis Menten curves for Fpg were generated by incubating probe OGR1 (0.03-0.3 μM) with 6 nM Fpg in Fpg reaction buffer and 100 μg/mL BSA at 37 °C and measuring the initial velocity at 37 °C. .. The initial velocity was determined by measuring the rate of fluorescence increase at 460 nm over the first 6 minutes of the reaction, during which the fluorescence increase was linear.

    Article Title: Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats
    Article Snippet: Duplex DNA substrates containing a single 8oxoG opposite of C, A, G, T, 5-hydroxycytosine (5OHC): G, 5-hydroxyuracil (5OHU): G, DHU: G, U: A or U: G base pair were generated by 32 P-5′ end-labeling of oligonucleotides using T4 polynucleotide kinase (New England Biolabs) as described previously ( ). .. The enzyme activities were assayed in a reaction buffer containing 70 mM 3-(N -morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 5% glycerol, and incubated at 37 °C for 1 h. Fpg Ec (New England Biolabs) was included as the positive control.

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. After electrophoresis, DNA was vacuum transferred to a nylon membrane (Roche Diagnostics, Mannheim, Germany) and hybridized with a PCR-generated probe to the corresponding region of mtDNA.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines. .. After electrophoresis and DNA transfer to a nylon membrane (Roche Diagnostics, Sigma, St. Louis, MO), portions of the membrane with DNA sequences of interest were hybridized with PCR-generated probes to the corresponding regions of mtDNA.

    other:

    Article Title: Human Alkyladenine DNA Glycosylase Employs a Processive Search for DNA Damage
    Article Snippet: E. coli formamidopyrimidine DNA glycosylase (FPG) was obtained from New England Biolabs.

    Article Title: Genotoxicity, cytotoxicity and gene expression in patients undergoing elective surgery under isoflurane anaesthesia
    Article Snippet: Ethidium bromide, HEPES and bovine serum albumin were purchased from Sigma (St Louis, MO, USA); normal and low melting point agaroses, EDTA and Tris from Invitrogen (Carlsbad, CA, USA); hydrogen peroxide (H2 O2 ), H3 BO3 , NaCl, NaOH, KCl, HCl, NaHCO3 , KH2 PO4 and Na2 HPO4 from Merck (Germany); Ficoll–Paque® from GE (Sweden); dimethylsulfoxide from Mallinckrodt (Mexico); Triton X-100 from J. T. Baker (Phillipsburg, NJ, USA); annexin labelled with fluorescein isothiocyanate (FITC) and annexin V buffer, 7-amino-actinomycin D (7-AAD), phycoerythrin (PE)-labelled monoclonal antibodies anti-hCD4+ and anti-hCD8+ were purchased from Becton Dickinson (San Jose, CA, USA) and endonuclease III (endo III) and formamidopyrimidine DNA glycosylase (Fpg) from New England Biolabs (Ipswich, MA, USA).

    Sequencing:

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. After electrophoresis, DNA was vacuum transferred to a nylon membrane (Roche Diagnostics, Mannheim, Germany) and hybridized with a PCR-generated probe to the corresponding region of mtDNA.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: This resulted in cutting mtDNA into two fragments – a small (2.7 kb) sequence containing the D-loop region and a large (13.6 kb) coding sequence. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines.

    Nucleic Acid Electrophoresis:

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: After 1 h lysis, the slides were placed on a horizontal gel electrophoresis unit filled with a fresh electrophoretic buffer (1 mM Na2 EDTA (sodium ethylenediamine tetraacetate) and 300 mM NaOH) and allowed to stay in the buffer for 40 min for DNA unwinding. .. The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in .

    Fluorescence:

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. Electrophoresis was performed and the slides dehydrated in 100% ethanol for 5 minutes before staining with ethidium bromide (10 ng/ml).

    Irradiation:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: The AP-lyase activity cleaves 3′ to the AP site leaving a 5′ phosphate and a 3′ ring opened sugar. .. 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 ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    Isolation:

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: Total DNA was isolated from lung samples powdered with a mortar and pestle, by previously described methods ( , ). .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: Before isolation all buffers were purged with nitrogen to prevent DNA oxidation. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines.

    Alkaline Single Cell Gel Electrophoresis:

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: Paragraph title: Alkaline comet assay ... The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in .

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: Paragraph title: Alkaline comet assay: primary and oxidative DNA damage ... For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Microscopy:

    Article Title:
    Article Snippet: Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA. .. Electrophoresis was then performed at 25 volts for 15 min, and the slides were dehydrated in 100% ethanol for 5 min, and then stained with ethidium bromide (10 ng/ml).

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. Electrophoresis was performed and the slides dehydrated in 100% ethanol for 5 minutes before staining with ethidium bromide (10 ng/ml).

    Purification:

    Article Title: Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats
    Article Snippet: DNA glycosylase reactions were performed by mixing purified protein as indicated, with 10–50 fmol DNA substrate in a total volume of 15 μL. .. The enzyme activities were assayed in a reaction buffer containing 70 mM 3-(N -morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 5% glycerol, and incubated at 37 °C for 1 h. Fpg Ec (New England Biolabs) was included as the positive control.

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: Purified DNA samples were digested with Ppu MI and Ahd I restriction enzymes (New England Biolabs, Beverly, MA) and used for further analyses. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: In brief, purified DNA was digested with Ppu MI and Ahd I restriction enzymes (New England Biolabs, Beverly, MA), 10 U/ μg DNA, overnight at 37 °C. .. To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines.

    Article Title: Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage
    Article Snippet: The cells were then lysed, and chromosomal DNA was extracted and purified as previously described ( ). .. To detect the formation of 8-oxo-G, 5 μg of DNA was digested with 14 units of formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) (New England, Biolabs, Ontario, Canada); this enzyme, in addition to its DNA glycosylase activity, possesses AP-lyase activity that generates single-strand breaks following 8-oxo-G attack ( ).

    Polymerase Chain Reaction:

    Article Title: Perinuclear Mitochondrial Clustering Creates an Oxidant-Rich Nuclear Domain Required for Hypoxia-Induced Transcription
    Article Snippet: The height as well as the pseudo-color of the intensity lines representing each pixel of the cell creates a visual map of ROS distribution throughout the cell. .. Hypoxia-induced oxidative base modifications in selected HRE sequences were detected with a previously described PCR-based technique wherein the bacterial DNA glycosylase Fpg (New England Biolabs) was applied to recognize and cleave 8-oxoguanine and related oxidized base products ( , ). .. Treatment of DNA with Fpg results in strand cleavage at sites of oxidized purines, thereby creating single-strand breaks that block PCR amplification.

    Article Title: Age-Associated Oxidative Damage to the p62 Promoter: Implications for Alzheimer's Disease
    Article Snippet: In brief, the formamidopyrimidine glycosylase (fpg) (New England Biolabs, Ipswich, MA) cleavage reaction was performed by incubating 250 ng of total genomic DNA with 8 units of fpg and 100 μg/ml of BSA in a total volume of 50 μl at 37°C for 12 hours, followed by incubation at 60°C for 10 min. to inactivate fpg. .. An aliquot of the reaction mixture was used for quantitative PCR assay.

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. Subsequently, Fpg-treated and untreated samples were incubated with 0.1 N NaOH for 15 min at 37°C, mixed with loading dye, and resolved in 0.6% agarose alkaline gel.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines. .. Samples containing 500 ng DNA were treated with 8 units of Fpg in 20 μl of reaction volume at 37 °C for 1 h. Subsequently, Fpg-treated and untreated samples were incubated with 0.1 N NaOH for 15 min at 37°C, and resolved in 0.6% agarose alkaline gel.

    Labeling:

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. After electrophoresis, DNA was vacuum transferred to a nylon membrane (Roche Diagnostics, Mannheim, Germany) and hybridized with a PCR-generated probe to the corresponding region of mtDNA.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines. .. After electrophoresis and DNA transfer to a nylon membrane (Roche Diagnostics, Sigma, St. Louis, MO), portions of the membrane with DNA sequences of interest were hybridized with PCR-generated probes to the corresponding regions of mtDNA.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2
    Article Snippet: The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH). .. The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH).

    Staining:

    Article Title: Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2
    Article Snippet: The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH). .. The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH).

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: After electrophoresis, the slides were washed with 0.4 M Tris, pH 7.5 (3 × 5 min) and stained with DAPI (4′,6-diamidino-2-phenylindole), 50 µL per slide (1 μg/mL). .. The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in .

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes.

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: After neutralization and dehydration, slides were air-dried before being stained with propidium iodide (PI). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Software:

    Article Title:
    Article Snippet: Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA. .. Electrophoresis was then performed at 25 volts for 15 min, and the slides were dehydrated in 100% ethanol for 5 min, and then stained with ethidium bromide (10 ng/ml).

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. Electrophoresis was performed and the slides dehydrated in 100% ethanol for 5 minutes before staining with ethidium bromide (10 ng/ml).

    Electrophoresis:

    Article Title: Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2
    Article Snippet: The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH). .. The units were hermetically closed, placed in a pre-heated moist box and incubated for 30 min at 37°C.

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Following T4pdg treatment, samples were loaded into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: After electrophoresis, the slides were washed with 0.4 M Tris, pH 7.5 (3 × 5 min) and stained with DAPI (4′,6-diamidino-2-phenylindole), 50 µL per slide (1 μg/mL). .. The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in .

    Article Title: Mitochondrial DNA integrity may be a determinant of endothelial barrier properties in oxidant-challenged rat lungs
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks. .. Subsequently, Fpg-treated and untreated samples were incubated with 0.1 N NaOH for 15 min at 37°C, mixed with loading dye, and resolved in 0.6% agarose alkaline gel.

    Article Title: Regulation of mitochondrial genome replication by hypoxia: the role of DNA oxidation in D-loop region
    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase (Fpg; New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines. .. Samples containing 500 ng DNA were treated with 8 units of Fpg in 20 μl of reaction volume at 37 °C for 1 h. Subsequently, Fpg-treated and untreated samples were incubated with 0.1 N NaOH for 15 min at 37°C, and resolved in 0.6% agarose alkaline gel.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes. .. For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes.

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1. .. Samples were then divided in two and digested or mock digested overnight with 10 units T4pdg [1× T4pdg reaction buffer, 100 µg/ml bovine serum albumin (BSA)].

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: After denaturation, which occurred in a solution comprising 300 mM NaOH and 1 mM EDTA in MilliQ water, slides underwent electrophoresis by setting constant voltage (25 V) and variable amperage (300 to 315 mA) for 20 min at 4 °C. .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Negative Control:

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: As a negative control, cells were incubated in LHC9 medium alone, while as a positive control cells were exposed (5 min at 4 °C, protected from light) to 110 μM hydrogen peroxide (H2 O2 ). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C.

    Agarose Gel Electrophoresis:

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Following T4pdg treatment, samples were loaded into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. For endonuclease treatment of MB + VL-damaged DNA, the EcoRI-digested samples were divided in half and incubated with or without 16 units of Fpg (1× NEB buffer 1, 100 µg/ml BSA) for 5 h. Following incubation, the samples were loaded directly into a 0.8% alkaline agarose gel and separated by electrophoresis (20–24 h at 10 V). .. Ad DNA was separated on denaturing 8% alkaline agarose gels run at 10 V for 20–24 h. Following gel electrophoresis, two 30-min washes in neutralisation buffer [1.5 M Tris (pH 7.4), 1.5 M NaCl] were performed after which DNA was transferred to a neutral nylon membrane (Hybond N, Amersham) by upward capillary transfer of 10× SSC.

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine
    Article Snippet: Prior to treatment of Ad DNA with either T4 pyrimidine-DNA glycosylase [T4pdg; New England Biolabs (NEB) M0308S] or formamidopyrimidine (Fapy)-DNA glycosylase (Fpg; NEB M02040), all samples were digested overnight by 40 units of EcoRI (NEB R0101) in a total reaction volume of 50 µl in 1× NEB buffer 1. .. Samples were then divided in two and digested or mock digested overnight with 10 units T4pdg [1× T4pdg reaction buffer, 100 µg/ml bovine serum albumin (BSA)].

    In Vitro:

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Quantitation Assay:

    Article Title: Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage
    Article Snippet: Paragraph title: Detection and quantitation of oxidative-stress-induced DNA damage. ... To detect the formation of 8-oxo-G, 5 μg of DNA was digested with 14 units of formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) (New England, Biolabs, Ontario, Canada); this enzyme, in addition to its DNA glycosylase activity, possesses AP-lyase activity that generates single-strand breaks following 8-oxo-G attack ( ).

    End Labeling:

    Article Title: Characterization of the major formamidopyrimidine-DNA glycosylase homolog in Mycobacterium tuberculosis and its linkage to variable tandem repeats
    Article Snippet: Duplex DNA substrates containing a single 8oxoG opposite of C, A, G, T, 5-hydroxycytosine (5OHC): G, 5-hydroxyuracil (5OHU): G, DHU: G, U: A or U: G base pair were generated by 32 P-5′ end-labeling of oligonucleotides using T4 polynucleotide kinase (New England Biolabs) as described previously ( ). .. The enzyme activities were assayed in a reaction buffer containing 70 mM 3-(N -morpholino) propane sulfonic acid, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol and 5% glycerol, and incubated at 37 °C for 1 h. Fpg Ec (New England Biolabs) was included as the positive control.

    Lysis:

    Article Title: Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2
    Article Snippet: After lysis, FPG and buffer slides were washed three times with cold enzyme buffer (40 mM HEPES, 0.1 M KCL, 0.5 mM EDTA, 0.2 mg/mL BSA, pH 8) before being clamped into slide units (12-Gel Comet Assay Unit™ , Severn Biotech Limited). .. The units were placed on ice and gels were respectively treated with either 30 μL enzyme buffer or 30 μL FPG solution (1:3000 dilution; FPG, New England Biolabs GmbH).

    Article Title: Crucial role of chelatable iron in silver nanoparticles induced DNA damage and cytotoxicity
    Article Snippet: After 1 h lysis, the slides were placed on a horizontal gel electrophoresis unit filled with a fresh electrophoretic buffer (1 mM Na2 EDTA (sodium ethylenediamine tetraacetate) and 300 mM NaOH) and allowed to stay in the buffer for 40 min for DNA unwinding. .. The treated cells were incubated on slides with the formamido-pyrimidine DNA glycosylase (FPG, New England BioLabs, UK), as described in .

    Article Title:
    Article Snippet: The coverslip was removed and the slide incubated in lysis buffer (2.5 m NaCl, 100 m m EDTA, 10 m m Tris, and 1% Triton X-100) for at least 4 h (or overnight). .. Each slide was then incubated with 8 units (in 100 μl volume) of formamidopyrimidine DNA glycosylase (FPG) (New England Biolabs) at 37 °C for 1 h. The FPG-treated slides were rinsed with Alkali buffer (300 m m NaOH and 1 m m EDTA; pH 12.1) for 20 min to denature DNA.

    Article Title: Modulation of DNA base excision repair during neuronal differentiation
    Article Snippet: The slide was incubated in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton X-100) for at least 4 hours then washed with neutralization buffer (0.4 M Tris, pH 7.4), followed by a 10 minute incubation with REC buffer (10 mM HEPES-KOH, 100 mM KCl, 10 mM EDTA, 0.1 mg/ml BSA; pH 7.4). .. For enzymatic treatments, slides were incubated with formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs) or mouse 3-methyladenine DNA glycosylase (Aag, Trevigen) at 37°C for 60 minutes.

    Article Title: Poorly soluble cobalt oxide particles trigger genotoxicity via multiple pathways
    Article Snippet: As a negative control, cells were incubated in LHC9 medium alone, while as a positive control cells were exposed (5 min at 4 °C, protected from light) to 110 μM hydrogen peroxide (H2 O2 ). .. For the analysis of oxidative DNA damage, after cell membrane lysis, the enzymes, formamidopyrimidine DNA glycosylase (FPG; New England Biolabs, Evry, France) and human 8-oxoguanine DNA N-glycosylase 1 (hOGG1; New England Biolabs, Evry, France), were added to the slides and incubated for 30 min at 37 °C. .. Slides, which were prepared in duplicate for each experimental condition, were analyzed under a fluorescence microscope at 400 X magnification using the Komet 6.0 software (Andor Bioimaging, Nottingham, UK).

    Activity Assay:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: The AP-lyase activity cleaves 3′ to the AP site leaving a 5′ phosphate and a 3′ ring opened sugar. .. 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 ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Article Title: Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage
    Article Snippet: DNA was quantified using the GeneQuant Pro program (Amersham Biosciences, Pittsburg, PA). .. To detect the formation of 8-oxo-G, 5 μg of DNA was digested with 14 units of formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) (New England, Biolabs, Ontario, Canada); this enzyme, in addition to its DNA glycosylase activity, possesses AP-lyase activity that generates single-strand breaks following 8-oxo-G attack ( ). .. Subsequently, the DNA was maintained in a denatured state and electrophoresed through 0.8% alkaline agarose gels with buffer recirculation, as described previously ( ).

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    New England Biolabs e coli fpg
    ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by <t>hAAG</t> (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) <t>FPG:</t> 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.
    E Coli Fpg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

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

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: ARP discriminates between AP sites generated by enzymatic and nonenzymatic release of N7-meG. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by spontaneous N7-meG depurination (nonenzymatic, blue triangles), or N7-meG excision by hAAG (enzymatic, red squares). Substrates were incubated either with purified proteins [( A ) FPG: 0.5 nM; ( C ) ARP: 10 nM] or Arabidopsis cell-free extracts [( B ) arp −/− : 8 µg; ( D ) fpg −/− : 8 µg]. Reactions for detection of AP endonuclease activity ( C and D ) were supplemented with 2 mM MgCl 2 . After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Article Snippet: DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Fluorescence

    FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

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

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: FPG incises AP sites generated by spontaneous depurination of N7-meG. ( A ) Double-stranded oligonucleotide substrates (20 nM) containing a single lesion opposite C (N7-meG, me-FAPy-G, or AP site generated by uracil excision) were incubated for 2 h at 37 °C with Arabidopsis FPG (10 nM), hAPE1 (1 U), hAAG (2 U), or E. coli Fpg (8 U). ( B ) A double-stranded oligonucleotide substrate containing a single N7-meG:C pair was preincubated in DNA incision assay buffer ( Methods ) at 37 °C for the indicated times and then treated with human APE1 (1 U), E. coli Fpg (8 U), or Arabidopsis FPG (10 nM) for 1 h at 37 °C, or with NaOH (15 mM) for 10 min. at 70 °C. After stabilization with NaBH 4, reaction products were separated by denaturing PAGE and detected by fluorescence scanning. Values are means with SEs from three independent experiments.

    Article Snippet: DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence

    Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.

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

    Article Title: Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis

    doi: 10.1073/pnas.1719497115

    Figure Lengend Snippet: Depurinated N7-meG is repaired through FPG-dependent SP-BER. DNA substrates (20 nM) were a 9:1 mixture of homoduplex G:C and heteroduplex AP:C generated either by uracil excision (enzymatic AP:C, Left ) or by spontaneous N7-meG depurination (nonenzymatic AP:C, Right ). Similar concentrations of each type of AP site were verified by incision with hAPE1 (10 U) (lanes 2 and 17). Substrates were incubated with Arabidopsis cell-free extracts (8 µg) for 1 h at 37 °C with or without either dGTP or all four dNTPs. After stabilization with NaBH 4 , reaction products were separated by denaturing PAGE and detected by fluorescence scanning. N.E., nonextract.

    Article Snippet: DNA substrates (20 nM) containing N7-meG or me-FAPy-G were validated in reactions (50 µL) with hAAG (2 U; NEB), hAPE1 (1 U; NEB), or E. coli Fpg (EcoFpg, 8 U; NEB).

    Techniques: Generated, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence

    The level of MutM-recognized DNA modifications in plasmid DNA. (A) RP4 DNA (positive control), treated in vitro with methylene blue (MB) and light, can be subsequently nicked in vitro with the MutM (Fpg) enzyme, indicating the presence of 8-oxo-guanine

    Journal:

    Article Title: The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

    doi: 10.1128/JB.00776-07

    Figure Lengend Snippet: The level of MutM-recognized DNA modifications in plasmid DNA. (A) RP4 DNA (positive control), treated in vitro with methylene blue (MB) and light, can be subsequently nicked in vitro with the MutM (Fpg) enzyme, indicating the presence of 8-oxo-guanine

    Article Snippet: The Fpg (also named MutM) enzyme was purchased from New England Biolabs (NEB).

    Techniques: Plasmid Preparation, Positive Control, In Vitro

    High copy numbers of mutM + and mutY + , as well as other potential defects in 8-oxo-dGTP interception, do not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM + or mutY + genes on the viability

    Journal:

    Article Title: The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

    doi: 10.1128/JB.00776-07

    Figure Lengend Snippet: High copy numbers of mutM + and mutY + , as well as other potential defects in 8-oxo-dGTP interception, do not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM + or mutY + genes on the viability

    Article Snippet: The Fpg (also named MutM) enzyme was purchased from New England Biolabs (NEB).

    Techniques:

    High copy number of mutM + and mutY + does not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM + or mutY + genes on the viability of mutT and recBC mutant strains. A total of 10 μl

    Journal:

    Article Title: The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

    doi: 10.1128/JB.00776-07

    Figure Lengend Snippet: High copy number of mutM + and mutY + does not kill mutT rec mutants. (A) The effect of high-copy-number plasmids carrying the mutM + or mutY + genes on the viability of mutT and recBC mutant strains. A total of 10 μl

    Article Snippet: The Fpg (also named MutM) enzyme was purchased from New England Biolabs (NEB).

    Techniques: Mutagenesis

    (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

    Journal:

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

    doi: 10.1016/j.freeradbiomed.2008.09.038

    Figure Lengend 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

    Article Snippet: The basis of the assay is that treatment of DNA with Fpg (New England Biolabs, Beverly, MA) results in strand cleavage at sites of oxidized purines, thereby creating single strand breaks that block PCR amplification.

    Techniques: 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

    Journal:

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

    doi: 10.1016/j.freeradbiomed.2008.09.038

    Figure Lengend 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

    Article Snippet: The basis of the assay is that treatment of DNA with Fpg (New England Biolabs, Beverly, MA) results in strand cleavage at sites of oxidized purines, thereby creating single strand breaks that block PCR amplification.

    Techniques: 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

    Journal:

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

    doi: 10.1016/j.freeradbiomed.2008.09.038

    Figure Lengend 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

    Article Snippet: The basis of the assay is that treatment of DNA with Fpg (New England Biolabs, Beverly, MA) results in strand cleavage at sites of oxidized purines, thereby creating single strand breaks that block PCR amplification.

    Techniques: Southern Blot, Polymerase Chain Reaction

    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.

    Journal: PLoS ONE

    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

    doi: 10.1371/journal.pone.0158581

    Figure Lengend 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.

    Article Snippet: 250 ng of each construct were digested with Formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs, Cat. #M0240S) using 1 μL of Fpg (8 units) in the presence of BSA, according to the manufacturer’s instructions, in 1X NEBuffer 1 for 1 hr at 37°C.

    Techniques: Construct, Purification, Negative Control, Positive Control