Htopo 1

Manufactured by TopoGEN

Sourced in United States

The HTopo I is a laboratory instrument designed for the analysis and manipulation of DNA topological structures. It provides a precise and controlled environment for studying the topological properties of DNA molecules.

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Lab products found in correlation

Htopo 2, by TopoGEN (2 mentions) Myecl imager, by Thermo Fisher Scientific (1 mentions) Compounds ranhc 5 and 6, by TopoGEN (1 mentions)

4 protocols using htopo 1

Topoisomerase I and II Inhibition Assays

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hTopoI relaxation assays were done using the supercoiled pHOT1 as substrate, with the recombinant hTopoI (TopoGEN, Port Orange, FL, USA) and compounds RANHC-V and -VI or DMSO (CTRL) following the manufacturer’s protocol (TopoGEN, Port Orange, FL, USA), with some changes [47 (link)].
Similarly, hTopoII decatenation assays were carried out incubating the kinetoplast DNA (kDNA) substrate with the hTopoII (TopoGEN, Port Orange, FL) and compounds RANHC-V and -VI or DMSO (CTRL) following the manufacturer’s procedures (TopoGEN, Port Orange, FL, USA), with some changes [47 (link)].

Ceramella J., Troiano R., Iacopetta D., Mariconda A., Pellegrino M., Catalano A., Saturnino C., Aquaro S., Sinicropi M.S, & Longo P. (2023). Synthesis of Novel N-Heterocyclic Carbene-Ruthenium (II) Complexes, “Precious” Tools with Antibacterial, Anticancer and Antioxidant Properties. Antibiotics, 12(4), 693.

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Topoisomerase Relaxation and Decatenation Assays

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hTopo I relaxation assays were performed by incubating the substrate (supercoiled pHOT1) with the recombinant hTopo I (TopoGEN, Port Orange, FL, USA) and compounds, as indicated in the supplier protocol (TopoGEN, Port Orange, FL, USA), with some revisions [80 (link)]. hTopo II decatenation assays were conducted by incubating the kinetoplast DNA substrate (kDNA) with the hTopo II (TopoGEN, Port Orange, FL) and studied compounds, as indicated in the supplier procedures (TopoGEN, Port Orange, FL, USA), with some changes as in [80 (link)].

Sinicropi M.S., Ceramella J., Vanelle P., Iacopetta D., Rosano C., Khoumeri O., Abdelmohsen S., Abdelhady W, & El-Kashef H. (2023). Novel Thiazolidine-2,4-dione-trimethoxybenzene-thiazole Hybrids as Human Topoisomerases Inhibitors. Pharmaceuticals, 16(7), 946.

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Topoisomerase I Relaxation Assay

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Calf Topo I (cTopo I) purchased from Thermo Fisher Scientific and hTopo I purchased from Topogen were used in this study. One unit of Topo I was defined as the amount of topoisomerase required to completely relax 0.3 μg of the negatively-supercoiled pBR322 plasmid DNA under the conditions described below.
Relaxation reaction mixtures (20 μL) contained 50 mM Tris-HCl (pH 7.5 at 23°C), 100 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol (DTT), 50 μg/ml bovine serum albumin (BSA), 0.3 μg of the supercoiled plasmid DNA, 1 unit of either human or calf Topo I, and the various concentrations of fluoroquinolones (11 ). Reaction mixtures were incubated at 37°C for 15 minutes and terminated by adding ethylenediaminetetraacetic acid (EDTA) to 25 mM. The DNA products were analyzed by electrophoresis through vertical 1.2% agarose gels at 2 V/cm for 15 hours in TAE buffer. Gels were stained with 0.5 μg/ml ethidium bromide, and then photographed and quantified using a MyECL Imager (Thermo Fisher Scientific).

Oppegard L.M., Delgado J.L., Kulkarni C.A., Towle T.R., Hart D.E., Williams B.P., Lentz S.R., Norris B.J., Flory C.M., Schumacher R.J., Murry D.J., Kerns R.J, & Hiasa H. (2018). Novel N-1 Substituted Fluoroquinolones Inhibit Human Topoisomerase I Activity and Exhibit Anti-proliferative Activity. Investigational new drugs, 37(2), 378-383.

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Topoisomerase I Relaxation Assay

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hTopo I relaxation assays have been performed in a final volume of 20 mL, as reported by Iacopetta et al. [37 (link),78 (link)], incubating 0.25 mg of supercoiled plasmid pHOT1 in Tris-EDTA (TE) buffer (TopoGEN, Port Orange, FL, USA) with tested compounds and recombinant hTopo I (2 units) (TopoGEN, Port Orange, FL, USA) for 1h at 37 °C. The obtained solution has been loaded in a 1% agarose gel containing 1× Tris-acetate-EDTA (TAE) buffer without ethidium bromide (EB). Agarose gel has been stained using 1× TAE buffer with EB (0.5 mg/mL) for 30 min and after washed with distilled water for 15 min. At the end, it has been visualized by using a ultraviolet (UV) transilluminator. The experiment was performed in triplicate.

Iacopetta D., Rosano C., Sirignano M., Mariconda A., Ceramella J., Ponassi M., Saturnino C., Sinicropi M.S, & Longo P. (2020). Is the Way to Fight Cancer Paved with Gold? Metal-Based Carbene Complexes with Multiple and Fascinating Biological Features. Pharmaceuticals, 13(5), 91.

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