These HR/NHEJ kits are cell based, meaning they have the HR or NHEJ GFP reporters pre-installed. A single plasmid (I-Sce1) is transfected to activate repair. Please see this table for a summary and comparison/application of each kit.
Transfection-based HR and NHEJ kits use transient transfections to make a pool of cells containing the DNA reporters to allow the investigator to interrogate the 2 major DNA repair pathways. The advantages are:
It uses GFP readout to measure the efficiency of repair.
There is zero background when repair is blocked or inhibited
The reporters for HR or NHEJ can be installed in virtually any cell line that can be transiently transfect
It is rapid and robust
It is ideal for drug screens to find novel inhibitors of HR/NHEJ
The kits are highly specific and can only report HR or NHEJ
Readout GFP can be done by FACS or Imaging
Entire pathway can be followed by live imaging in viable cells
Ideal to track repair kinetics at the single cell level
High purity, catalytically active Top1 Enzyme overexpressed in the baculovirus system and purified to single band homogeneity. These preparations are cheaper than native topo I made from human tissue and offer excellent purity and high specific activity.
If you want to order this product, please contact us.
High purity, catalytically active Human Topoisomerase I Enzyme over-expressed in the baculovirus system and purified to single band homogeneity. These preparations are cheaper than native Topo I made from human tissue and offer excellent purity and high specific activity. In addition, the 100 kDa form of recombinant topo I is stable and is less prone to proteolysis, relative to the native protein from human cells.
The final preparation of Topo 1 is highly active in relaxation of plasmid DNA.
Titration of Human Topoisomerase I Enzyme Activity
Topo I as prepared and purified using the baculovirus overexpression system. Activity was determined by serial 2 fold dilutions using Supercoiled DNA. The data show that this preparation titrates to approximately 8 units/ul. One unit of Human Topo I can relax >50% of 0.25 μg (250 ng) of supercoiled plasmid DNA in 30 minutes at 37°C.
Included Materials
10X assay buffer and dilution buffer is included. These may be purchased separately as TGS 10X Topo I Assay Buffer (TG4095).
Quality Control Tests
1. Human Topo I is certified to be nuclease free.
2. A check for cross contamination with topo II was negative. There was no decatenation of KDNA in topo II reaction conditions.
3. The final fraction of topo I is a column pool and is in the following buffer: 20mM NaH2PO4 (pH7.4), 300mM NaCl, 50ug/ml BSA, 50% glycerol, 50mM Imidazole. The final fraction was analyzed by SDS-PAGE and shown to contain a single, predominant band of 100 kDa.
4. Activity assays. Relaxation assays were carried out in a final volume of 25 μl in top1 reaction buffer (10X reaction buffer–also called TGS–supplied with this product is: 100 mM Tris-Cl (pH 7.9), 10 mM EDTA, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol). Supercoiled plasmid DNA was included at 0.25 μg/reaction. Reactions are terminated with 5 μl (per 20 μl reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 1% native agarose gel (no ethidium bromide in the gel or buffer during electrophoresis).
Human Topoisomerase I enzyme is shipped on dry ice and should be stored at -20°C. It has a life-time of about 6 months when stored under optimal conditions. Repeat freeze/thaw cycles will accelerate loss.
Yoshizaki A, Yanaba K, Ogawa A, Asano Y, Kadono T, Sato S: Immunization With DNA Topoisomerase I and Freund’s Complete Adjuvant Induces Skin and Lung Fibrosis and Autoimmunity via Interleukin-6 Signaling.Arthritis and Rheumatism 2011, 63: 3575-3585. doi: 10.1002/art.30539.
Zhang L, Sullivan P, Suyama J, et. al: Epidermal growth factor-induced heparanase nucleolar localization augments DNA topoisomerase I activity in brain metastatic breast cancer.Molecular Cancer Research 2010, 8: 278-290. doi: 10.1158/1541-7786.MCR-09-0375
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