|Products||Human Topoisomerase II, Alpha from USB Corp.|
|Item||Human Topoisomerase II, Alpha|
|Description|| Source: E. coli containing a clone of the human Topoisomerase II gene. |
Description:Topoisomerase II alters the topological state of nucleic acids by passing an intact DNA helix through a transient break which generates a separate DNA helix(1,2). As a result of its double-stranded DNA passage mechanism, the enzyme can relax negatively or positively supercoiled DNA, as well as catenate/decatenate or knot/unknot DNA molecules. Topoisomerase II has an absolute requirement for divalent cation and ATP (or dATP).
Tested User Friendly
Molecular Weight: 340,000 Da homodimer
Optimum Temperature: 3037C
Optimum pH: 7.9, rapidly loses activity below pH 7.5
Requirements for Divalent Cation: 5mM Mg2+ is optimum for catalytic activity and 5mM Ca2+
for DNA cleavage.
Optimum ATP Concentration: 0.5 to 1.0mM
Optimum Ion Strength: 100-170mM KCl/NaCl
Inhibitors: N-ethylmaleimide, zinc, novobicin, coumermycin A1, etoposide, amsacrine
Purity:Greater than 98% pure as determined by silver-stained SDS-PAGE. Free of contaminating exonucleases and endonucleases.
Storage Buffer:15mM sodium phosphate (pH 7.1), 700mM NaCl, 0.1mM EDTA, 0.5mM DTT, 50% glycerol.
Shipping and Storage:
Shipped on dry ice. Store at -20C for frequent use. Store at -80C for long-term storage.
Assay Conditions:The reaction mixture contains 10mM Tris-HCl, pH 7.9, 175mM KCl, 0.1mM EDTA, 5mM MgCl2, 2.5% glycerol, 0.5mM ATP.
Unit Definition:One unit is the amount of enzyme required to fully relax 0.3 g (5nM) of negatively supercoiled pBR322 plasmid DNA in 30 minutes at 37C under the standard assay conditions.
Tested User FriendlyTM Functional Test: Tested for activity in standard Topoisomerase II unit assay.
Applications:*Relaxing negatively or positively supercoiled DNA.
*Catenating or decatenating DNA.
*Knotting or unknotting DNA.
PROTOCOL FOR RELAXATION OF SUPERCOILED DNA:
Topoisomerase II, Alpha and 10X Topoisomerase II Reaction Buffer (PN 73592) are functionally tested in the following protocol using 300 ng (0.1 pmol) pBR322 DNA at 30C.
10X Topo II Reaction Buffer 2 l
Supercoiled DNA 0.11.0 pmol
Topoisomerase II, Alpha 110 units
H2O to 20 l
*Mix and incubate at 3037C for 15 minutes.
*Stop the reaction by the addition of 3 l of 7mM EDTA or by the addition of SDS
|Info|| USB Corp.|
26111 Miles Road
Cleveland Ohio 44128
Fax Number: 216.464.5075
Web Site: http://www.usbweb.com