end  (New England Biolabs)

Journal: International Journal of Molecular Sciences

Article Title: The Protective Effect of Dabigatran and Rivaroxaban on DNA Oxidative Changes in a Model of Vascular Endothelial Damage with Oxidized Cholesterol

doi: 10.3390/ijms21061953

Figure Lengend Snippet: The level of DNA pyrimidine and purine oxidation in HUVECs (analysis by alkaline comet assay using endonuclease III (Nth)) or human 8 oxoguanine DNA glycosylase (hOOG1) was induced by 25-hydroxycholesterol (10 µg/mL) ( A ). The repair of DNA damage in cells was induced by ( B ) dabigatran (100 ng/mL and 500 ng/mL) and ( C ) rivaroxaban (100 ng/mL and 500 ng/mL). Each experiment included a positive control (PC) which concerned the cells incubated with hydrogen peroxide at 20 µM for 15 min on ice and subsequently treated with the enzymes. The value of DNA in the figure captions for comet tail in the presence of either enzyme for all groups was reduced by the value obtained in the comet assay without any enzyme and the value for enzyme buffer only. The number of cells scored from each slide was 100. The mean value for 100 cells analyzed in each treatment in four independent experiments (400 total cells) was recorded. Mean ± SEM was calculated from four individual experiments (400 comets) and was significantly different from negative controls at * p

Article Snippet: Endonuclease III (Nth) and human 8-oxoguanine DNA glycosylase (hOGG1) were acquired in New England Biolabs (Ipswich, MA, USA).

Techniques: Alkaline Single Cell Gel Electrophoresis, Positive Control, Incubation, Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

doi: 10.1093/nar/gkab817

Figure Lengend Snippet: DNA damaging potential and effect of antioxidants in isolated PBMCs. ( A ) Illustration of sample collection and treatment: 1. PBMCs isolated from blood samples taken from healthy volunteers; 2. isolated cells treated with Cu(II)–TC1 in the presence or absence of ROS scavengers (pyruvate, tiron, L-histidine, D-mannitol). ( B ) Schematic of DNA labelling process: 3. DNA with damaged bases (purple) was extracted from PBMCs; 4. BER enzymes (Fpg, Endo III or APE1, cyan) remove oxidized bases; 5. fluorescent dNTPs (aminoallyl-dUTP-ATTO-647N, red) were incorporated by DNA polymerase 1 (grey); 6. dsDNA was labelled with YOYO-1 (green). ( C ) Representative microscopic images of labelled DNA from non-treated PBMCs (top) and Cu(II)–TC1 treated cells (bottom, Cu(II):TC1 ratio 1:1, 300:300 μM). Scale bar = 10 μm. Extended panel shown in Supplementary Figure S8 . ( D ) DNA damage at varying TC1 concentrations with fixed Cu(II) concentration with and without radical scavengers (Cu(II):TC1 ratio 4:1, 300:75 μM). ( E ) Identification of specific DNA damage lesions with individual BER enzymes (Cu(II):TC1 ratio 4:1, 300:75 μM). Enzyme and scavenger controls are shown in Supplementary Figure S9 .

Article Snippet: CutSmart buffers, pUC19 plasmid (N3041) and repair enzymes—Fpg (M0240S), Endo III (M0268S), Endo IV (M0304S), Endo V (M0305S), hAAG (M0313S) and APE1 (M0282S)—were purchased from New England Biolabs.

Techniques: Isolation, Concentration Assay

Journal: BioMed Research International

Article Title: Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage

doi: 10.1155/2013/587493

Figure Lengend Snippet: Characterization of digested BAC vector and PFGE size fractionation of digested HMW by Hind III. (a) Characterization of digested vector by electrophoresis; M: 1 kb DNA ladder; 1: BAC vector digested by Hind III; 2: BAC vector digested by B am H I and X ho I; (b) initial PFGE size fractionation of partially digested HMW; M: Lamda Ladder PFG Marker; 1, 5, 10, 15, 20, and 50 U/ μ gDNA, respectively; (c) second-size selection PFGE, used to eliminate smaller DNA molecules; M: Lamda Ladder PFG Marker; 100–200 kb, 200–300 kb, 300–400 kb, respectively; (d) quantification of Wuzhishan miniature pig genomic DNA.1: 100–200 kb DNA; 2: 200–300 kb DNA; 3: 300–400 kb DNA.

Article Snippet: After establishing the minimal amount of restriction enzyme required for complete digestion of the vector in a series of test reactions, the vector DNA was digested with an appropriate amount of Hind III restriction endonuclease (New England Biolabs) and treated with alkaline phosphatase (CIAP) for dephosphorylation [ ].

Techniques: BAC Assay, Plasmid Preparation, Fractionation, Electrophoresis, Marker, Selection

Journal: PLoS ONE

Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

doi: 10.1371/journal.pone.0157270

Figure Lengend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

Article Snippet: Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

Techniques: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection