LAE Biotech LAE Biotech LAE Biotech

Human Topoisomerase I, active

(Catalog # P031)



Human topoisomerase I is produced from a baculovirus expression system, purified from freshly extracted cell lysates, and purified to homogeneity using methods developed by LAE Biotechnology Co., LTD. The final fraction of enzyme contains a single polypeptide band of 100 kDa and is devoid of contaminating nuclease activity.



l          Studying the effects of supercoiling on transcription in vitro.

l          Studying chromatin reconstitution in vitro.

l          Determining the degree of supercoiling of naturally occurring DNA.

l          Detecting mutant plasmids that differ in length by only one basepair.

l          Increasing restriction endonuclease digestion of resistant DNA substrates by ¡§unwinding¡¨ the DNA coils to expose restriction sites.

l          Anticancer drug screening.


Unit Definition:

A unit of topo I can relax 0.2 ug pGEM 5Z DNA in 30 min at 37oC. The enzyme is supplied at a nominal concentration of approximately 2 units/ul.


Quality Control Tests:

1. A test for nuclease contamination is carried out by assaying for the formation of linear KDNA and linear plasmid DNA. Incubation with KDNA or supercoiled pGEM 5Z DNA (4 hrs. at 37oC in the presence of 10 mM MgCl2) is performed. Linear DNA or breakdown products are not generated under these conditions.

2. A check for cross contamination with Topo II is negative. There must be no decatenation of KDNA in topo II reaction conditions.


Storage Buffer:

The final fraction of topoisomerase I is an affinity column pool and is in the following buffer: 10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 100 mM imidazole, 0.5 mM PMSF, 1 mM DTT, 10 % glycerol. The enzyme is stable in this buffer at -70oC. In general the enzyme is less stable when diluted. We recommend that you do not dilute the enzyme before storage at -70oC. In addition, it is a good practice to aliquot the enzyme after the first thaw.


Dilution Buffer:

Dilutions should be performed in 1x Reaction Buffer (recipe given below).


Assay Conditions:

Relaxation assays are carried out in a final volume of 10-20 ul in topo I reaction buffer (40 mM Tris-Cl, pH 7.5, 100 mM NaCl or KCl, 10mM MgCl2, 0.5 mM EDTA, 30 ug/ml BSA). Assays: Supercoiled plasmid DNA is used at 0.2 ug/reaction. Reactions are terminated with 5 ul (per 20 ul reaction volume) of stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25% glycerol). Reaction products are analyzed on a 0.8% native agarose gel.


5X Reaction Buffer (cat# B001) [200 mM Tris-HCl (pH 7.5), 500 mM KCl, 50 mM MgCl2, 50 mM DTT, 2.5 mM EDTA and 150 ug/ml BSA].


Quality Control:

This product has passed the following quality control assays: absence of detectable endodeoxyribonuclease, exodeoxyribonuclease, and phosphatase activities; performance in converting super-coiled DNA to relaxed DNA.



The enzyme is shipped on ice and must be stored at -20 degree or lower.



Wang, J.C. (1996) Annu. Rev. Biochem. 65: 635-692.


        Comassiae blue staining of purified hTOP1

        Left: Coomassie Blue staining of purified human DNA topoisomerase protein.

Right: A unit of topo I incubated with 0.2 ug pGEM 5Z DNA in 30 min at 37oC.