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Trevigen fpg
Increased oxidative <t>DNA</t> damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with <t>Fpg</t> and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).
Fpg, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells"

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0090261

Increased oxidative DNA damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with Fpg and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).
Figure Legend Snippet: Increased oxidative DNA damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with Fpg and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).

Techniques Used: DNA Extraction, Real-time Polymerase Chain Reaction, Amplification

Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24 HPRT gene using LA-QPCR. (A) left: NEIL2 knockdown from hPF was tested by qRT-PCR; right: Western blots showing shRNA-mediated knockdown of NEIL2 in HEK293 cells expressing Flag-NEIL2. (B) Cultured hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control followed by DNA isolation and digestion with Fpg and Nei as described in Materials and Methods. The DNA was used as template for HPRT large and short fragment amplification by LA-QPCR assay. The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL 2 knockdown were statistically significant (P
Figure Legend Snippet: Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24 HPRT gene using LA-QPCR. (A) left: NEIL2 knockdown from hPF was tested by qRT-PCR; right: Western blots showing shRNA-mediated knockdown of NEIL2 in HEK293 cells expressing Flag-NEIL2. (B) Cultured hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control followed by DNA isolation and digestion with Fpg and Nei as described in Materials and Methods. The DNA was used as template for HPRT large and short fragment amplification by LA-QPCR assay. The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL 2 knockdown were statistically significant (P

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, shRNA, Expressing, Cell Culture, DNA Extraction, Amplification

Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24h in the human POLB gene using LA-QPCR. (A) Cultured primary hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control on the left, followed by processing of the DNA with Fpg and Nei. The DNA was used for Polβ ( POLB ) large and short fragment amplification by LA-QPCR. Quantification was with ImageQuant (Molecular Dynamics). The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL2 knockdown were statistically significant (P
Figure Legend Snippet: Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24h in the human POLB gene using LA-QPCR. (A) Cultured primary hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control on the left, followed by processing of the DNA with Fpg and Nei. The DNA was used for Polβ ( POLB ) large and short fragment amplification by LA-QPCR. Quantification was with ImageQuant (Molecular Dynamics). The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL2 knockdown were statistically significant (P

Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Amplification

2) Product Images from "Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage"

Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage

Journal: Journal of Nucleic Acids

doi: 10.1155/2017/8154646

The effect of Fen-1 knockdown. (a) On repair of oxidative DNA damage. Top: western analysis of Fen-1 protein in AGS cells transfected with pLKO-1 sh Fen-1 to knock down Fen-1 or control vector; levels of PCNA protein were used as a loading control. Bottom: comet assay. AGS cells were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay. (b) On DNA replication. S-phase cells were marked.
Figure Legend Snippet: The effect of Fen-1 knockdown. (a) On repair of oxidative DNA damage. Top: western analysis of Fen-1 protein in AGS cells transfected with pLKO-1 sh Fen-1 to knock down Fen-1 or control vector; levels of PCNA protein were used as a loading control. Bottom: comet assay. AGS cells were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay. (b) On DNA replication. S-phase cells were marked.

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Single Cell Gel Electrophoresis

The effect of PCNA knockdown on the repair of oxidative DNA damage. (a) Comet assay. AGS cells transfected with pLKO-1 shPCNA to knock down PCNA or control vector were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay to monitor the levels of H 2 O 2 -induced DNA lesions in cells. Bottom panel: western analysis of PCNA protein with levels of β -actin protein as a loading control. (b) Immunostaining analysis and (c) ELISA. Cells transfected with pLKO-1 shPCNA or control vector were treated with 1 mM H 2 O 2 for 1 and 4 h before being harvested for immunofluorescence and ELISA assays of 8-oxo-dG. Typical images were shown. N for control, that is, cells without treatment. ∗∗ for p
Figure Legend Snippet: The effect of PCNA knockdown on the repair of oxidative DNA damage. (a) Comet assay. AGS cells transfected with pLKO-1 shPCNA to knock down PCNA or control vector were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay to monitor the levels of H 2 O 2 -induced DNA lesions in cells. Bottom panel: western analysis of PCNA protein with levels of β -actin protein as a loading control. (b) Immunostaining analysis and (c) ELISA. Cells transfected with pLKO-1 shPCNA or control vector were treated with 1 mM H 2 O 2 for 1 and 4 h before being harvested for immunofluorescence and ELISA assays of 8-oxo-dG. Typical images were shown. N for control, that is, cells without treatment. ∗∗ for p

Techniques Used: Single Cell Gel Electrophoresis, Transfection, Plasmid Preparation, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, Immunofluorescence

3) Product Images from "Guanine-specific DNA damage induced by ?-irradiated histone"

Article Title: Guanine-specific DNA damage induced by ?-irradiated histone

Journal:

doi: 10.1042/BJ20050186

Effect of Fpg treatment on DNA damage induced by histone H1-hydroperoxides in the presence of Cu(I) Reaction mixtures contained the 32 P-5′-end-labelled 337 bp fragment, calf thymus DNA (20 μM in DNA bases), the indicated concentrations of histone H1-hydroperoxides and 20 μM Cu(I) in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. The mixture was incubated at 37 °C for 60 min. The DNA fragments were then treated with Fpg and electrophoresed on an 8% polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel.
Figure Legend Snippet: Effect of Fpg treatment on DNA damage induced by histone H1-hydroperoxides in the presence of Cu(I) Reaction mixtures contained the 32 P-5′-end-labelled 337 bp fragment, calf thymus DNA (20 μM in DNA bases), the indicated concentrations of histone H1-hydroperoxides and 20 μM Cu(I) in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. The mixture was incubated at 37 °C for 60 min. The DNA fragments were then treated with Fpg and electrophoresed on an 8% polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel.

Techniques Used: Incubation

Autoradiogram of 32 P-labelled DNA fragments incubated with various histone protein-hydroperoxides in the presence of Cu(I) The reaction mixtures contained the 32 P-5′-end-labelled 337 bp DNA fragment, calf thymus DNA (20 μM in DNA bases), 0.7 μM of histone protein-hydroperoxides (histone H1, H2A, H3 and H4) and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. After incubation at 37 °C for 60 min, DNA fragments were treated with 10 units of Fpg protein at 37 °C for 120 min. The DNA fragments were electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel. Oligonucleotides generated by hydroperoxide-induced DNA damage are indicated by arrows. The bands above these oligonucleotides are single- and double-strand intact DNA fragments.
Figure Legend Snippet: Autoradiogram of 32 P-labelled DNA fragments incubated with various histone protein-hydroperoxides in the presence of Cu(I) The reaction mixtures contained the 32 P-5′-end-labelled 337 bp DNA fragment, calf thymus DNA (20 μM in DNA bases), 0.7 μM of histone protein-hydroperoxides (histone H1, H2A, H3 and H4) and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. After incubation at 37 °C for 60 min, DNA fragments were treated with 10 units of Fpg protein at 37 °C for 120 min. The DNA fragments were electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel. Oligonucleotides generated by hydroperoxide-induced DNA damage are indicated by arrows. The bands above these oligonucleotides are single- and double-strand intact DNA fragments.

Techniques Used: Incubation, Generated

4) Product Images from "Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny"

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny

Journal: PLoS ONE

doi: 10.1371/journal.pone.0203863

SSB, ALS and 8oxoG repair of UVA-irradiated KSCs and TAs (at 10 J/cm 2 ) using comet assay +- Fpg/T4endoV. The results are expressed as the median % of remaining lesions. The results for 8oxoG and TT-dimers were obtained by subtraction of comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD. Statistical analyses were performed for each population (dotted line for TAs and black line for KSCs) between each time and the previous time; * p
Figure Legend Snippet: SSB, ALS and 8oxoG repair of UVA-irradiated KSCs and TAs (at 10 J/cm 2 ) using comet assay +- Fpg/T4endoV. The results are expressed as the median % of remaining lesions. The results for 8oxoG and TT-dimers were obtained by subtraction of comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD. Statistical analyses were performed for each population (dotted line for TAs and black line for KSCs) between each time and the previous time; * p

Techniques Used: Irradiation, Single Cell Gel Electrophoresis

SSB, ALS, 8oxoG and thymine dimer induction in UVA-irradiated KSCS and TAs (at 10 J/cm 2 ) by comet assay +- Fpg/T4 endoV. The results are expressed as the median % tail intensity. The results for 8oxoG and TT-dimers were obtained by subtraction of the comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD; * p
Figure Legend Snippet: SSB, ALS, 8oxoG and thymine dimer induction in UVA-irradiated KSCS and TAs (at 10 J/cm 2 ) by comet assay +- Fpg/T4 endoV. The results are expressed as the median % tail intensity. The results for 8oxoG and TT-dimers were obtained by subtraction of the comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD; * p

Techniques Used: Irradiation, Single Cell Gel Electrophoresis

5) Product Images from "DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease"

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease

Journal: Aging Cell

doi: 10.1111/acel.12541

Polβ deficiency impairs olfactory bulb formation and causes DNA damage in newly generated neurons, while not adversely affecting NPC proliferation or mitochondrial function. A. Images showing WT and Polβ‐deficient (HO) embryos and whole brain (E17). Polβ‐deficient embryos are smaller than WT embryos. Scale bar = 1 cm. The brains of Polβ‐deficient embryos are smaller than their WT counterparts and do not develop a discernable olfactory bulb (arrow). Scale bar = 2 mm. B. Images showing neurospheres, and neurons and astrocytes differentiated from neurospheres, in cultures established from WT and Polβ −/− embryos. Scale bar = 100 μm. C. Results of flow cytometry cell cycle analysis of NPCs from WT and Polβ −/− embryos. D. Results of flow cytometry‐based measurements of the indicated parameters: MMP, mitochondrial membrane potential; Mito. content, mitochondrial DNA content; cellular reactive oxygen species (ROS); and mitochondrial ROS. E. Results of alkali comet assay with formamidopyrimidine DNA glycosylase (Fpg) enzymatic digestion. Comet assay analysis revealed accumulation of oxidative DNA damage in Polβ‐deficient neurons. n ≥ 5. *** P
Figure Legend Snippet: Polβ deficiency impairs olfactory bulb formation and causes DNA damage in newly generated neurons, while not adversely affecting NPC proliferation or mitochondrial function. A. Images showing WT and Polβ‐deficient (HO) embryos and whole brain (E17). Polβ‐deficient embryos are smaller than WT embryos. Scale bar = 1 cm. The brains of Polβ‐deficient embryos are smaller than their WT counterparts and do not develop a discernable olfactory bulb (arrow). Scale bar = 2 mm. B. Images showing neurospheres, and neurons and astrocytes differentiated from neurospheres, in cultures established from WT and Polβ −/− embryos. Scale bar = 100 μm. C. Results of flow cytometry cell cycle analysis of NPCs from WT and Polβ −/− embryos. D. Results of flow cytometry‐based measurements of the indicated parameters: MMP, mitochondrial membrane potential; Mito. content, mitochondrial DNA content; cellular reactive oxygen species (ROS); and mitochondrial ROS. E. Results of alkali comet assay with formamidopyrimidine DNA glycosylase (Fpg) enzymatic digestion. Comet assay analysis revealed accumulation of oxidative DNA damage in Polβ‐deficient neurons. n ≥ 5. *** P

Techniques Used: Generated, Flow Cytometry, Cytometry, Cell Cycle Assay, Single Cell Gel Electrophoresis

6) Product Images from "Riboflavin activated by ultraviolet A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C"

Article Title: Riboflavin activated by ultraviolet A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C

Journal:

doi: 10.1073/pnas.0610534104

Quantification of induced DNA lesions. ( a ) DNA damage in the genome. Cleavage assay with Fpg digestion and alkaline gel electrophoresis of the genomic DNA of mouse embryonic fibroblasts treated with riboflavin or irradiated with UVA1, individually or
Figure Legend Snippet: Quantification of induced DNA lesions. ( a ) DNA damage in the genome. Cleavage assay with Fpg digestion and alkaline gel electrophoresis of the genomic DNA of mouse embryonic fibroblasts treated with riboflavin or irradiated with UVA1, individually or

Techniques Used: Cleavage Assay, Nucleic Acid Electrophoresis, Irradiation

7) Product Images from "Mitochondrial base excision repair assays"

Article Title: Mitochondrial base excision repair assays

Journal:

doi: 10.1016/j.ymeth.2010.02.020

Endogenous oxidative damage is overestimated in mtDNA due to oxidative lesions generated from the mtDNA purification process. Fpg/Southern blot analysis was used to measure 8-oxoG levels in nDNA as well as in mtDNA with and without mitochondrial isolation;
Figure Legend Snippet: Endogenous oxidative damage is overestimated in mtDNA due to oxidative lesions generated from the mtDNA purification process. Fpg/Southern blot analysis was used to measure 8-oxoG levels in nDNA as well as in mtDNA with and without mitochondrial isolation;

Techniques Used: Generated, Purification, Southern Blot, Isolation

Related Articles

Amplification:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

Blocking Assay:

Article Title: Mitochondrial base excision repair assays
Article Snippet: We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]). .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Electrophoresis:

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: The slides were washed three times in 0.4 M Tris pH 7.4 for 10 min and submersed in a horizontal chamber in electrophoresis buffer (0.3 M NaOH, 1 mM Na-EDTA) for 20 min at 4°C. .. For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells
Article Snippet: Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider. .. Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider.

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: To facilitate detection of oxidized guanines in addition to DNA strand breaks, slides were incubated with E. coli formamidopyrimidine-DNA glycosylase (Fpg) prior to electrophoresis. .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD).

Incubation:

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: The slides were neutralized three times for 10 min with Tris buffer (0.4 M pH 7.4), stained with 50 μl of GelRed and then incubated at 4°C in a light-tight wet chamber until analysis of the slides. .. For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

Article Title: Mitochondrial base excision repair assays
Article Snippet: Based on the above strategies we describe here a consensus protocol for detection and quantification of mtDNA 8-oxoG damage and repair. (1) Cells growing in covered slides are treated with oxidative damaging agent, such as hydrogen peroxide (e.g. 500μM for 30 minutes) or menadione (e.g. 50μM for 30 min), in serum free medium. (2) Then the medium is removed, replaced with complete medium, and incubated for repair times 0, 2, 4, 12, 24 h. Also include an untreated control slide. (3) After washing three times with cold PBS, the slides are fixed with cold methanol (we have also used 1:1 methanol-acetone) for 20 min at −20°C. (4) After rinsing slides in room temperature (RT) PBS two times, the slides are treated with RNase solution for 1 h at 37°C. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells
Article Snippet: DNA oxidative damage was identified via incubation with formamidopyrimidine DNA glycosylase (Fpg) to reveal 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, aflatoxin B1-fapy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil [ , ]. .. Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider.

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: In brief, conventional alkaline comet assay procedures were performed to obtain nucleoids in agarose gels on slides. .. Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. EndoIII and Fpg are bacterial glycosylases that specifically recognize oxidized pyrimidines and purines, respectively.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Fpg readily cleaves 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and converts resultant abasic sites into strand breaks, which are detectable by the alkaline comet assay. .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Subsequently slides were incubated 40 min in alkaline buffer (0.3 M NaOH, 1 mM EDTA), electrophoresed at 1.2 V/cm (300 mA, 25 V), washed 3 times in neutralization buffer (0.4 M Tris, pH 7.5) and stained with 20 µg/ml ethidium bromide.

Article Title: Direct and indirect roles of RECQL4 in modulating base excision repair capacity
Article Snippet: Cells were treated with 500 µM H2 O2 for 15 min and analyzed immediately or washed and incubated in fresh medium at 37°C and 5% CO2 for 3 h. Cells were washed with PBS and 300 µl of mincing solution [1× HBSS (Ca2+ , Mg2+ free), 20 m m EDTA, 10% DMSO) was added. .. Cells were treated with formamidopyrimidine-DNA glycosylase (Fpg) in FPG FLARE reaction buffer (1× FLARE, 1× BSA, Trevigen, Inc.) or buffer alone for 1 h at 37°C.

Activity Assay:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: This kit minimizes DNA oxidation during the isolation step and has been used previously for LA-QPCR assays . .. After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. To examine the formation of oxidative DNA damage in the human gene, hypoxanthine phosphoribosyl transferase 1 (HPRT ), the LA-QPCR assay was performed in SSS-exposed human cells as described previously , , .

Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells
Article Snippet: FPG is a glycolase that recognizes and specifically cuts the oxidised bases principally 8-oxoG from DNA, producing apurinic sites converted in breaks by the associated AP-endonuclease activity. .. The analysis was performed using a FPG FLARE module (Trevigen, Gaithersburg, MD).

Single Cell Gel Electrophoresis:

Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage
Article Snippet: Paragraph title: 2.4. Comet Assay with Enzymes ... To measure oxidative DNA adducts with the alkaline comet assay, we performed an additional step immediately after the cell lysis step, that is, incubating the slides containing the nucleoids with Endo III and Fpg (from Trevigen Co.; 2 units of each enzyme per slide in buffer with 10 mM Tris-HCl pH 7.4) as described previously [ ].

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: At least 50 comet images were acquired and analyzed for each group. .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C. .. Slides were then incubated in alkali solution (pH 12.1) for 30 min and placed in electrophoresis chamber filled with prechilled 1x TBE buffer and run for 30 min at 35 V. The DNA was then stained with ethidium bromide and viewed on a Zeiss Axiovert 200 M fluorescent microscope (Zeiss, Thornwood NY).

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD).

Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells
Article Snippet: Therefore, these breaks can be detected by comet assay and give a measure of oxidative DNA damage, enabling us to detect moderate, but still appreciable damage. .. The analysis was performed using a FPG FLARE module (Trevigen, Gaithersburg, MD).

Article Title: Direct and indirect roles of RECQL4 in modulating base excision repair capacity
Article Snippet: Paragraph title: Comet assay ... Cells were treated with formamidopyrimidine-DNA glycosylase (Fpg) in FPG FLARE reaction buffer (1× FLARE, 1× BSA, Trevigen, Inc.) or buffer alone for 1 h at 37°C.

Modification:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide.

Countercurrent Chromatography:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

Transfection:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide.

Footprinting:

Article Title: Riboflavin activated by ultraviolet A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C
Article Snippet: Alternatively, the DNA digest can be analyzed by footprinting methodologies, e.g., LM-PCR, enabling the detection of lesions at the level of nucleotide resolution ( ). .. Briefly, DNA was digested with an excess amount of Fpg (Trevigen, Gaithersburg, MD) or T4 Endo V (Epicentre, Madison, WI) in special buffers supplied by the respective manufacturers.

other:

Article Title: Guanine-specific DNA damage induced by ?-irradiated histone
Article Snippet: Nuclease P1 (400 units/mg) was purchased from Yamasa Shoyu (Chiba, Japan), Fpg ( Escherichia coli formamidopyrimidine-DNA glycosylase) from Trevigen (Gaithersburg, MD, U.S.A.) and histone H1 (subgroup f1-lysine-rich fraction, calf thymus) and other histone proteins from Sigma (St. Louis, MO, U.S.A.).

Article Title: Long-range oxidative damage in duplex DNA: the effect of bulged G in a G-C tract and tandem G/A mispairs
Article Snippet: The Fpg (Trevigen Corporation; used as received) reactions were performed in 20 µl of solution that contained 50 mM Tris–HCl (pH 7.5), 2.0 mM EDTA, 100 mM NaCl and 5.0 µM DNA at 37°C for 1 h. A 15.6 ng portion of Fpg was used in each experiment.

Imaging:

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C. .. Slides were then incubated in alkali solution (pH 12.1) for 30 min and placed in electrophoresis chamber filled with prechilled 1x TBE buffer and run for 30 min at 35 V. The DNA was then stained with ethidium bromide and viewed on a Zeiss Axiovert 200 M fluorescent microscope (Zeiss, Thornwood NY).

Article Title: Direct and indirect roles of RECQL4 in modulating base excision repair capacity
Article Snippet: Cells were treated with formamidopyrimidine-DNA glycosylase (Fpg) in FPG FLARE reaction buffer (1× FLARE, 1× BSA, Trevigen, Inc.) or buffer alone for 1 h at 37°C. .. Slides were incubated in alkali solution (pH 12.1) for 30 min and then placed in a horizontal electrophoresis chamber filled with pre-chilled 1× TBE and run for 30 min at 35 V. DNA was stained with ethidium bromide and viewed on a Zeiss Axiovert 200 M fluorescence microscope (Zeiss, Thornwood NY).

Article Title: Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells
Article Snippet: Briefly, DNA was digested with an excess amount of UVDE (Trevigen, Gaithersburg, MD), T4 Endo V (Epicentre, Madison, WI), or Fpg (Trevigen) in buffers supplied by the manufacturers. .. The electrophoretic profiles were determined using standard ethidium bromide staining.

Polymerase Chain Reaction:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. LongAmp Taq DNA polymerase (New England Biolabs, Beverly, MA) was used to amplify a 10.4 kb region of HPRT in human genomic DNA using the following primers: 5′-TGG GAT TAC ACG TGT GAA CCA ACC-3′ and 5′-GCT CTA CCC TCT CCT CTA CCG TCC-3′ .

DNA Extraction:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After exposure to SSS-treated medium (0.1, 0.2 and 0.4 PE dilutions) at 37°C for 24 h as described above, the cells were harvested for genomic DNA extraction using the Qiagen Genomic-tip 20/G kit (Qiagen, Valencia, CA) per the manufacturer's instructions. .. After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

Nucleic Acid Electrophoresis:

Article Title: Riboflavin activated by ultraviolet A1 irradiation induces oxidative DNA damage-mediated mutations inhibited by vitamin C
Article Snippet: The digested DNA is then subjected to alkaline gel electrophoresis for assessment of damage in the overall genome. .. Briefly, DNA was digested with an excess amount of Fpg (Trevigen, Gaithersburg, MD) or T4 Endo V (Epicentre, Madison, WI) in special buffers supplied by the respective manufacturers.

Article Title: Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells
Article Snippet: The enzymatic digestion assays are based on the recognition and cleavage of specific types of DNA damage by specialized DNA repair enzymes, followed by gel electrophoresis. .. Briefly, DNA was digested with an excess amount of UVDE (Trevigen, Gaithersburg, MD), T4 Endo V (Epicentre, Madison, WI), or Fpg (Trevigen) in buffers supplied by the manufacturers.

Fluorescence:

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: Nuclear comets were imaged using a fluorescence microscope and analyzed using comet analysis software. .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C.

Article Title: Mitochondrial base excision repair assays
Article Snippet: [ ] used conventional fluorescence microscopy, and found that they can discriminate 8-oxoG staining in mtDNA from that in nDNA based on the difference in denaturing conditions required (likely because the differences in DNA packaging): for detection in mtDNA, pretreatment with NaOH was ideal, whereas denaturing with HCl resulted in a nDNA signal. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. After enzyme digestion, DNA in nucleoids was alkaline denatured and then separated with electrophoresis.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Subsequently slides were incubated 40 min in alkaline buffer (0.3 M NaOH, 1 mM EDTA), electrophoresed at 1.2 V/cm (300 mA, 25 V), washed 3 times in neutralization buffer (0.4 M Tris, pH 7.5) and stained with 20 µg/ml ethidium bromide.

Isolation:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: This kit minimizes DNA oxidation during the isolation step and has been used previously for LA-QPCR assays . .. After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Briefly, cells isolated from cerebrum were counted, mixed with low melting agarose, spread onto multiple slides for each time point (2×104 cells per slide), and incubated 16 h at 4°C in alkaline cell lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris pH 10 [set with NaOH], 1% Triton X-100). .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD).

Labeling:

Article Title: Mitochondrial base excision repair assays
Article Snippet: [ ] demonstrated simultaneous staining of nuclear and mitochondrial 8-oxoG in cultures cells, using confocal microscopy combined with mitotracker labeling to discriminate the subcellular compartments. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Mouse Assay:

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C. .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Comet tail moments for individual nuclei were determined using the CASP comet analysis software [ ] at .

Microscopy:

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: A total of 100 mL of the cell suspension mixture was spread on a microscope slide coated with 1% agarose and chilled to ice temperature. .. For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: Nuclear comets were imaged using a fluorescence microscope and analyzed using comet analysis software. .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C.

Article Title: Mitochondrial base excision repair assays
Article Snippet: [ ] used conventional fluorescence microscopy, and found that they can discriminate 8-oxoG staining in mtDNA from that in nDNA based on the difference in denaturing conditions required (likely because the differences in DNA packaging): for detection in mtDNA, pretreatment with NaOH was ideal, whereas denaturing with HCl resulted in a nDNA signal. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells
Article Snippet: Briefly, after treatment, the cells immersed in LMP agarose 1% were layered on microscope slides precoated with 1% normal melting point agarose and immersed in lysis buffer for at least 1 h at 4°C. .. Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider.

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. After enzyme digestion, DNA in nucleoids was alkaline denatured and then separated with electrophoresis.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Subsequently slides were incubated 40 min in alkaline buffer (0.3 M NaOH, 1 mM EDTA), electrophoresed at 1.2 V/cm (300 mA, 25 V), washed 3 times in neutralization buffer (0.4 M Tris, pH 7.5) and stained with 20 µg/ml ethidium bromide.

Confocal Microscopy:

Article Title: Mitochondrial base excision repair assays
Article Snippet: [ ] demonstrated simultaneous staining of nuclear and mitochondrial 8-oxoG in cultures cells, using confocal microscopy combined with mitotracker labeling to discriminate the subcellular compartments. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Lysis:

Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage
Article Snippet: Data are presented as averages of at least three independent experiments ± standard error. .. To measure oxidative DNA adducts with the alkaline comet assay, we performed an additional step immediately after the cell lysis step, that is, incubating the slides containing the nucleoids with Endo III and Fpg (from Trevigen Co.; 2 units of each enzyme per slide in buffer with 10 mM Tris-HCl pH 7.4) as described previously [ ]. .. Endo III and Fpg are bacterial glycosylases that specifically recognize oxidized pyrimidines and purines, respectively.

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: The slides were neutralized three times for 10 min with Tris buffer (0.4 M pH 7.4), stained with 50 μl of GelRed and then incubated at 4°C in a light-tight wet chamber until analysis of the slides. .. For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C. .. The slides were stained with GelRed, and comet analysis was performed using the image analysis Komet 6.0 software (Kinetic Imaging Ltd., Andor Technology plc., Belfast, Ireland).

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells
Article Snippet: Briefly, after treatment, the cells immersed in LMP agarose 1% were layered on microscope slides precoated with 1% normal melting point agarose and immersed in lysis buffer for at least 1 h at 4°C. .. Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Briefly, cells isolated from cerebrum were counted, mixed with low melting agarose, spread onto multiple slides for each time point (2×104 cells per slide), and incubated 16 h at 4°C in alkaline cell lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris pH 10 [set with NaOH], 1% Triton X-100). .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD).

Chloramphenicol Acetyltransferase Assay:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. Preliminary assays were carried out to ensure the linearity of PCR amplification with respect to the number of cycles and DNA concentration.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Fpg readily cleaves 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and converts resultant abasic sites into strand breaks, which are detectable by the alkaline comet assay. .. Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat4040-100-01, Gaithersburg, Trevigen, MD). .. Subsequently slides were incubated 40 min in alkaline buffer (0.3 M NaOH, 1 mM EDTA), electrophoresed at 1.2 V/cm (300 mA, 25 V), washed 3 times in neutralization buffer (0.4 M Tris, pH 7.5) and stained with 20 µg/ml ethidium bromide.

Plasmid Preparation:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide. .. DNA was transferred to nylon membrane, and blots were processed with an AlkPhos Direct Labeling and Detection Kit (GE Healthcare) using a probe against the f1 origin.

Software:

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: Nuclear comets were imaged using a fluorescence microscope and analyzed using comet analysis software. .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C.

Article Title: Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells
Article Snippet: Alternatively, the OTM score obtained by the software was employed. .. Oxidative lesions were digested by Fpg (Trevigen, CA, USA), under ideal conditions of pH and temperature suggested by the provider.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD). .. Slides were viewed using 20× objective on IX71 Olympus fluorescence microscope and images of nuclei captured with QIC-F-M-12-C cooled digital camera.

Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells
Article Snippet: The analysis was performed using a FPG FLARE module (Trevigen, Gaithersburg, MD). .. Slides were analysed using a fluorescence microscope (Leica) equipped with a camera.

Article Title: Direct and indirect roles of RECQL4 in modulating base excision repair capacity
Article Snippet: Cells were treated with formamidopyrimidine-DNA glycosylase (Fpg) in FPG FLARE reaction buffer (1× FLARE, 1× BSA, Trevigen, Inc.) or buffer alone for 1 h at 37°C. .. Slides were incubated in alkali solution (pH 12.1) for 30 min and then placed in a horizontal electrophoresis chamber filled with pre-chilled 1× TBE and run for 30 min at 35 V. DNA was stained with ethidium bromide and viewed on a Zeiss Axiovert 200 M fluorescence microscope (Zeiss, Thornwood NY).

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide. .. DNA was transferred to nylon membrane, and blots were processed with an AlkPhos Direct Labeling and Detection Kit (GE Healthcare) using a probe against the f1 origin.

Alkaline Single Cell Gel Electrophoresis:

Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage
Article Snippet: Data are presented as averages of at least three independent experiments ± standard error. .. To measure oxidative DNA adducts with the alkaline comet assay, we performed an additional step immediately after the cell lysis step, that is, incubating the slides containing the nucleoids with Endo III and Fpg (from Trevigen Co.; 2 units of each enzyme per slide in buffer with 10 mM Tris-HCl pH 7.4) as described previously [ ]. .. Endo III and Fpg are bacterial glycosylases that specifically recognize oxidized pyrimidines and purines, respectively.

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: Paragraph title: Alkaline comet assay ... For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: In brief, conventional alkaline comet assay procedures were performed to obtain nucleoids in agarose gels on slides. .. Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber.

Article Title: TRANSGENIC OVEREXPRESSION OF NEUROGLOBIN ATTENUATES FORMATION OF SMOKE INHALATION-INDUCED OXIDATIVE DNA DAMAGE, IN VIVO, IN THE MOUSE BRAIN
Article Snippet: Paragraph title: Alkaline Comet assay ... Accordingly, duplicate slide sets were used: all slides were equilibrated with the Fpg reaction buffer (10 mM HEPES-KOH, 100 mM KCl, 0.1 mg/ml BSA, pH 7.4) and then incubated either with or without Fpg (cat#4040-100-01, Gaithersburg, Trevigen, MD).

Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells
Article Snippet: Paragraph title: Standard and FPG-modified alkaline comet assay ... The analysis was performed using a FPG FLARE module (Trevigen, Gaithersburg, MD).

Agarose Gel Electrophoresis:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide. .. DNA was transferred to nylon membrane, and blots were processed with an AlkPhos Direct Labeling and Detection Kit (GE Healthcare) using a probe against the f1 origin.

Concentration Assay:

Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells
Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity. .. LongAmp Taq DNA polymerase (New England Biolabs, Beverly, MA) was used to amplify a 10.4 kb region of HPRT in human genomic DNA using the following primers: 5′-TGG GAT TAC ACG TGT GAA CCA ACC-3′ and 5′-GCT CTA CCC TCT CCT CTA CCG TCC-3′ .

Construct:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide.

Alkaline Lysis:

Article Title: 8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells
Article Snippet: Cells were transfected with the 8OG-containing construct, and the plasmid was reisolated at various times after transfection with a modified alkaline lysis protocol, as previously described ( , ). .. Plasmid was digested with 1U Fpg (Trevigen) for 2 h at 37 °C and resolved on a 0.6% agarose gel containing 5 μg/ml ethidium bromide.

Migration:

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. Nucleoids after staining with propidium iodide were examined with a fluorescence microscope; images of at least 50 nucleoids per slide were recorded with a closed-circuit display CCD camera (Zeiss/Axioskop 2 Mot plus).

Staining:

Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny
Article Snippet: The slides were neutralized three times for 10 min with Tris buffer (0.4 M pH 7.4), stained with 50 μl of GelRed and then incubated at 4°C in a light-tight wet chamber until analysis of the slides. .. For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

Article Title: DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease
Article Snippet: Nuclear DNA was electrophoresed at 25 V for 30 min at 4◦ C and stained with SYBR Gold (Life Technologies, MD). .. For assessing DNA damage in neuronal cells, we performed alkali comet assay on slides, where cells were additionally exposed to formamidopyrimidine DNA glycosylase (Fpg) diluted in FPG FLARE reaction buffer (1x FLARE, 1x BSA, Trevigen, MD) or buffer alone for 1 h at 37 °C.

Article Title: Mitochondrial base excision repair assays
Article Snippet: [ ] used conventional fluorescence microscopy, and found that they can discriminate 8-oxoG staining in mtDNA from that in nDNA based on the difference in denaturing conditions required (likely because the differences in DNA packaging): for detection in mtDNA, pretreatment with NaOH was ideal, whereas denaturing with HCl resulted in a nDNA signal. .. We have used 100 μg/ml of RNAase in Tris buffer (pH 7.5). (5) At this point one can perform a few 3 min washes in 1% Triton X-100 (in PBS), for cellular permeabilization, if desired (see [ ]), then wash and replace with PBS. (6) To confirm specificity of the 8-oxoG antibody, one can now incubate the slides with Fpg (we have used 1 U/μl in FLARE buffer (Trevigen) and) or MutM (10 μg/ml) or Dnase I (1 U/μl) for 1 hour at 37°C (see [ ]).

Article Title: Is the Oxidative DNA Damage Level of Human Lymphocyte Correlated with the Antioxidant Capacity of Serum or the Base Excision Repair Activity of Lymphocyte?
Article Snippet: Each slide was incubated with 2 units of both EndoIII and Fpg (Trevigen, Inc., Gaithersburg, MD, USA) in buffer of 10 mM Tris-HCl pH 7.4 for 1 h at 37°C in a sealed, humid chamber. .. After enzyme digestion, DNA in nucleoids was alkaline denatured and then separated with electrophoresis.

Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells
Article Snippet: At the end of treatment, before performing comet assay, an aliquot of cells from each sample was dyed with trypan blue in order to evaluate the percentage of cell death (blue stained cells). .. The analysis was performed using a FPG FLARE module (Trevigen, Gaithersburg, MD).

Article Title: Rapid repair of UVA-induced oxidized purines and persistence of UVB-induced dipyrimidine lesions determine the mutagenicity of sunlight in mouse cells
Article Snippet: Briefly, DNA was digested with an excess amount of UVDE (Trevigen, Gaithersburg, MD), T4 Endo V (Epicentre, Madison, WI), or Fpg (Trevigen) in buffers supplied by the manufacturers. .. After ethanol precipitation, the digests were loaded onto a 1.5% alkaline agarose gel, and run at 3.0 V/cm for 4 hours, with constant recirculation of the running buffer.

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    Trevigen fpg
    Increased oxidative <t>DNA</t> damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with <t>Fpg</t> and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).
    Fpg, supplied by Trevigen, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Increased oxidative DNA damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with Fpg and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).

    Journal: PLoS ONE

    Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

    doi: 10.1371/journal.pone.0090261

    Figure Lengend Snippet: Increased oxidative DNA damage in various human cells. Following exposure to a series of dilutions of SSS–laden DMEM for 24 h, DNA isolation and digestion with Fpg and Nei followed by qPCR for amplification of HPRT large and a short fragment (LA-QPCR assay). Three cell lines, HEK293, hPF and BEAS-2B, all exhibited a dose-response relationship between levels of oxidative DNA damage and the SSS doses used in the LA-QPCR assay. PE: puff equivalents (the smoke from one puff in 1 ml of medium).

    Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

    Techniques: DNA Extraction, Real-time Polymerase Chain Reaction, Amplification

    Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24 HPRT gene using LA-QPCR. (A) left: NEIL2 knockdown from hPF was tested by qRT-PCR; right: Western blots showing shRNA-mediated knockdown of NEIL2 in HEK293 cells expressing Flag-NEIL2. (B) Cultured hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control followed by DNA isolation and digestion with Fpg and Nei as described in Materials and Methods. The DNA was used as template for HPRT large and short fragment amplification by LA-QPCR assay. The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL 2 knockdown were statistically significant (P

    Journal: PLoS ONE

    Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

    doi: 10.1371/journal.pone.0090261

    Figure Lengend Snippet: Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24 HPRT gene using LA-QPCR. (A) left: NEIL2 knockdown from hPF was tested by qRT-PCR; right: Western blots showing shRNA-mediated knockdown of NEIL2 in HEK293 cells expressing Flag-NEIL2. (B) Cultured hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control followed by DNA isolation and digestion with Fpg and Nei as described in Materials and Methods. The DNA was used as template for HPRT large and short fragment amplification by LA-QPCR assay. The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL 2 knockdown were statistically significant (P

    Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

    Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, shRNA, Expressing, Cell Culture, DNA Extraction, Amplification

    Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24h in the human POLB gene using LA-QPCR. (A) Cultured primary hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control on the left, followed by processing of the DNA with Fpg and Nei. The DNA was used for Polβ ( POLB ) large and short fragment amplification by LA-QPCR. Quantification was with ImageQuant (Molecular Dynamics). The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL2 knockdown were statistically significant (P

    Journal: PLoS ONE

    Article Title: NEIL2 Protects against Oxidative DNA Damage Induced by Sidestream Smoke in Human Cells

    doi: 10.1371/journal.pone.0090261

    Figure Lengend Snippet: Effect of NEIL2 knockdown on induction of oxidative DNA damage by exposure to SSS for 24h in the human POLB gene using LA-QPCR. (A) Cultured primary hPF with NEIL2 knockdown were treated with 3 doses of SSS extract as compared to the control on the left, followed by processing of the DNA with Fpg and Nei. The DNA was used for Polβ ( POLB ) large and short fragment amplification by LA-QPCR. Quantification was with ImageQuant (Molecular Dynamics). The histogram depicts the mean % of DNA tail ± SEM of 3 to 4 independent experiments. The symbol * means that the differences between the Control and NEIL2 knockdown were statistically significant (P

    Article Snippet: After quantification equal amounts of genomic DNA were digested with two E. coli BER enzymes, Fpg (Trevigen, Gaithersburg, MD) and Nei (provided from Dr. Tapas lab), which are able to remove a variety of oxidized purine and pyrimidine oxidized DNA bases and induce strand breaks by cleaving the phosphodiester bond with their associated AP lyase activity.

    Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Amplification

    The effect of Fen-1 knockdown. (a) On repair of oxidative DNA damage. Top: western analysis of Fen-1 protein in AGS cells transfected with pLKO-1 sh Fen-1 to knock down Fen-1 or control vector; levels of PCNA protein were used as a loading control. Bottom: comet assay. AGS cells were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay. (b) On DNA replication. S-phase cells were marked.

    Journal: Journal of Nucleic Acids

    Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage

    doi: 10.1155/2017/8154646

    Figure Lengend Snippet: The effect of Fen-1 knockdown. (a) On repair of oxidative DNA damage. Top: western analysis of Fen-1 protein in AGS cells transfected with pLKO-1 sh Fen-1 to knock down Fen-1 or control vector; levels of PCNA protein were used as a loading control. Bottom: comet assay. AGS cells were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay. (b) On DNA replication. S-phase cells were marked.

    Article Snippet: To measure oxidative DNA adducts with the alkaline comet assay, we performed an additional step immediately after the cell lysis step, that is, incubating the slides containing the nucleoids with Endo III and Fpg (from Trevigen Co.; 2 units of each enzyme per slide in buffer with 10 mM Tris-HCl pH 7.4) as described previously [ ].

    Techniques: Western Blot, Transfection, Plasmid Preparation, Single Cell Gel Electrophoresis

    The effect of PCNA knockdown on the repair of oxidative DNA damage. (a) Comet assay. AGS cells transfected with pLKO-1 shPCNA to knock down PCNA or control vector were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay to monitor the levels of H 2 O 2 -induced DNA lesions in cells. Bottom panel: western analysis of PCNA protein with levels of β -actin protein as a loading control. (b) Immunostaining analysis and (c) ELISA. Cells transfected with pLKO-1 shPCNA or control vector were treated with 1 mM H 2 O 2 for 1 and 4 h before being harvested for immunofluorescence and ELISA assays of 8-oxo-dG. Typical images were shown. N for control, that is, cells without treatment. ∗∗ for p

    Journal: Journal of Nucleic Acids

    Article Title: Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage

    doi: 10.1155/2017/8154646

    Figure Lengend Snippet: The effect of PCNA knockdown on the repair of oxidative DNA damage. (a) Comet assay. AGS cells transfected with pLKO-1 shPCNA to knock down PCNA or control vector were treated with 20 μ M H 2 O 2 for indicated periods of time before being harvested for comet-Fpg/Endo III assay to monitor the levels of H 2 O 2 -induced DNA lesions in cells. Bottom panel: western analysis of PCNA protein with levels of β -actin protein as a loading control. (b) Immunostaining analysis and (c) ELISA. Cells transfected with pLKO-1 shPCNA or control vector were treated with 1 mM H 2 O 2 for 1 and 4 h before being harvested for immunofluorescence and ELISA assays of 8-oxo-dG. Typical images were shown. N for control, that is, cells without treatment. ∗∗ for p

    Article Snippet: To measure oxidative DNA adducts with the alkaline comet assay, we performed an additional step immediately after the cell lysis step, that is, incubating the slides containing the nucleoids with Endo III and Fpg (from Trevigen Co.; 2 units of each enzyme per slide in buffer with 10 mM Tris-HCl pH 7.4) as described previously [ ].

    Techniques: Single Cell Gel Electrophoresis, Transfection, Plasmid Preparation, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, Immunofluorescence

    Effect of Fpg treatment on DNA damage induced by histone H1-hydroperoxides in the presence of Cu(I) Reaction mixtures contained the 32 P-5′-end-labelled 337 bp fragment, calf thymus DNA (20 μM in DNA bases), the indicated concentrations of histone H1-hydroperoxides and 20 μM Cu(I) in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. The mixture was incubated at 37 °C for 60 min. The DNA fragments were then treated with Fpg and electrophoresed on an 8% polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel.

    Journal:

    Article Title: Guanine-specific DNA damage induced by ?-irradiated histone

    doi: 10.1042/BJ20050186

    Figure Lengend Snippet: Effect of Fpg treatment on DNA damage induced by histone H1-hydroperoxides in the presence of Cu(I) Reaction mixtures contained the 32 P-5′-end-labelled 337 bp fragment, calf thymus DNA (20 μM in DNA bases), the indicated concentrations of histone H1-hydroperoxides and 20 μM Cu(I) in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. The mixture was incubated at 37 °C for 60 min. The DNA fragments were then treated with Fpg and electrophoresed on an 8% polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel.

    Article Snippet: Nuclease P1 (400 units/mg) was purchased from Yamasa Shoyu (Chiba, Japan), Fpg ( Escherichia coli formamidopyrimidine-DNA glycosylase) from Trevigen (Gaithersburg, MD, U.S.A.) and histone H1 (subgroup f1-lysine-rich fraction, calf thymus) and other histone proteins from Sigma (St. Louis, MO, U.S.A.).

    Techniques: Incubation

    Autoradiogram of 32 P-labelled DNA fragments incubated with various histone protein-hydroperoxides in the presence of Cu(I) The reaction mixtures contained the 32 P-5′-end-labelled 337 bp DNA fragment, calf thymus DNA (20 μM in DNA bases), 0.7 μM of histone protein-hydroperoxides (histone H1, H2A, H3 and H4) and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. After incubation at 37 °C for 60 min, DNA fragments were treated with 10 units of Fpg protein at 37 °C for 120 min. The DNA fragments were electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel. Oligonucleotides generated by hydroperoxide-induced DNA damage are indicated by arrows. The bands above these oligonucleotides are single- and double-strand intact DNA fragments.

    Journal:

    Article Title: Guanine-specific DNA damage induced by ?-irradiated histone

    doi: 10.1042/BJ20050186

    Figure Lengend Snippet: Autoradiogram of 32 P-labelled DNA fragments incubated with various histone protein-hydroperoxides in the presence of Cu(I) The reaction mixtures contained the 32 P-5′-end-labelled 337 bp DNA fragment, calf thymus DNA (20 μM in DNA bases), 0.7 μM of histone protein-hydroperoxides (histone H1, H2A, H3 and H4) and 20 μM CuCl in 200 μl of 10 mM sodium phosphate buffer (pH 7.4) containing 5 μM DTPA. After incubation at 37 °C for 60 min, DNA fragments were treated with 10 units of Fpg protein at 37 °C for 120 min. The DNA fragments were electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel (12 cm×16 cm), and autoradiograms were obtained by exposing an X-ray film to the gel. Oligonucleotides generated by hydroperoxide-induced DNA damage are indicated by arrows. The bands above these oligonucleotides are single- and double-strand intact DNA fragments.

    Article Snippet: Nuclease P1 (400 units/mg) was purchased from Yamasa Shoyu (Chiba, Japan), Fpg ( Escherichia coli formamidopyrimidine-DNA glycosylase) from Trevigen (Gaithersburg, MD, U.S.A.) and histone H1 (subgroup f1-lysine-rich fraction, calf thymus) and other histone proteins from Sigma (St. Louis, MO, U.S.A.).

    Techniques: Incubation, Generated

    SSB, ALS and 8oxoG repair of UVA-irradiated KSCs and TAs (at 10 J/cm 2 ) using comet assay +- Fpg/T4endoV. The results are expressed as the median % of remaining lesions. The results for 8oxoG and TT-dimers were obtained by subtraction of comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD. Statistical analyses were performed for each population (dotted line for TAs and black line for KSCs) between each time and the previous time; * p

    Journal: PLoS ONE

    Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny

    doi: 10.1371/journal.pone.0203863

    Figure Lengend Snippet: SSB, ALS and 8oxoG repair of UVA-irradiated KSCs and TAs (at 10 J/cm 2 ) using comet assay +- Fpg/T4endoV. The results are expressed as the median % of remaining lesions. The results for 8oxoG and TT-dimers were obtained by subtraction of comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD. Statistical analyses were performed for each population (dotted line for TAs and black line for KSCs) between each time and the previous time; * p

    Article Snippet: For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

    Techniques: Irradiation, Single Cell Gel Electrophoresis

    SSB, ALS, 8oxoG and thymine dimer induction in UVA-irradiated KSCS and TAs (at 10 J/cm 2 ) by comet assay +- Fpg/T4 endoV. The results are expressed as the median % tail intensity. The results for 8oxoG and TT-dimers were obtained by subtraction of the comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD; * p

    Journal: PLoS ONE

    Article Title: Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny

    doi: 10.1371/journal.pone.0203863

    Figure Lengend Snippet: SSB, ALS, 8oxoG and thymine dimer induction in UVA-irradiated KSCS and TAs (at 10 J/cm 2 ) by comet assay +- Fpg/T4 endoV. The results are expressed as the median % tail intensity. The results for 8oxoG and TT-dimers were obtained by subtraction of the comet assay results with and without Fpg and T4endoV, respectively; the mean of samples from 3 donors +- SD; * p

    Article Snippet: For revealing oxidized purines and pyrimidine dimers, Fpg (Trevigen, Gaithersburg, MD, USA) and T4endoV (BioLabs, Ipswich, MA, USA) were added after the lysis step at 0.05 U/μL for 45 min at 37°C.

    Techniques: Irradiation, Single Cell Gel Electrophoresis