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Human
Topoisomerase I, active
(Catalog
# P031)
Description:
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.
Applications:
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.
Buffer:
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.
Shipping/Storage:
The
enzyme is shipped on ice and must be stored at
-20 degree or lower.
Reference:
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.
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