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Human Topoisomerase II Beta, active
(Catalog
# P033)
Description:
DNA topoisomerase IIb alters the topological state of nucleic acids by
passing an intact DNA helix through a transient break which generates
a separate DNA helix by changing linking number by 2 for each
reaction. As a result of its
double-stranded DNA passage mechanism, Topo IIb can relax negatively or positively supercoiled DNA,
as well as catenate/decatenate or knot/unknot DNA molecules. Topo IIb has an absolute requirement for divalent cation and
ATP. LAE human topoisomerase
IIb 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 180 kDa and is devoid of contaminating
nuclease activity.
Applications:
l. Studying the effects of supercoiling on transcription in
vitro.
2. Studying chromatin reconstitution in vitro.
3. Determining the degree of supercoiling of naturally occurring
DNA.
4. Anticancer drug screening.
Unit Definition:
A unit of topo IIb
can relax 0.2 ug pGEM 5Z DNA or KDNA 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 I
is negative. There must be no relaxation of supercoiling plasmid DNA
in the reaction conditions without ATP.
Storage Buffer:
The final fraction of topoisomerase IIb is an affinity column pool and is
in the following buffer: 10 mM Tris-Cl, pH 7.5, 500 mM NaCl, 500 mM
Imidazol, 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).
Buffer:
5X Reaction Buffer (cat# B003) [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.
Legal
consideration: FOR RESEARCH USE
ONLY.
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.
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