商品编号


TG2005H-RC2



品牌


TopoGEN



公司


TopoGEN



公司分类


Topoisomerases



Size

500 Units

商品信息


Human Topo I Product Background

TopoGEN
now offers a high purity and 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.? 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.


Quality Control Tests:


1

A test for nuclease contaxamination was carried out by assaying for the formation of linear KDNA and linear plasmid DNA.? Incubations of 1 ?g of catenated KDNA or supercoiled pUC19 DNA (4 hrs. at 37°C in the presence of 10 mM MgCl2) were performed.? Linear DNA or breakdown products were not generated under these conditions.



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 topoisomerase 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.
*
Actual concentration can range from 25mM-100mM, according to how stock enzyme was diluted in storage buffer.
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?


Activity Assays:
Relaxation assays were carried out in a final volume of 25 ?l in recombinant topo I reaction buffer (10X reaction buffer, supplied with this product is: 100 mM Tris-Cl, pH 7.9, 1.5 M NaCl, 1% BSA, 1 mM Spermidine, 50% glycerol).?? Supercoiled plasmid DNA was included at 0.25 ?g/reaction.? Reactions terminated with 5 ?l (per 20 ?l reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products were analyzed on a 1% native agarose gel.? Under these conditions, relaxation activity is easily detectable (typically we see 10-40 units of activity per ul).? A typical titration is shown below:
?





Reviews and Citations:

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



Included Materials:
10X assay buffer and dilution buffer is included.? These may be purchased separately as TGS 10X Topo I Assay Buffer (TG4095).