Purified Human Topoisomerase II Alpha (Top2a) overexpressed and purified to homogeneity to a single band on SDS-PAGE. Topoisomerase II is highly purified and free of nuclease contamination.
Human Topo II Quality Control Tests
Nuclease contamination: assayed by testing for linear kDNA and linear plasmid DNA formation. Incubation of 1 µg of catenated KDNA or supercoiled pUC19 DNA for 4 hrs. at 37° C (under top2a assay conditions and with or without ATP).
Cross contamination with topo I was assessed by assaying for relaxation of supercoiled DNA under conditions where Human Topoisomerase II Alpha activity is suppressed. Excess purified enzyme was incubated with pRYG DNA in the absence of ATP (with and without Mg++) for 2 hr at 37° C. Under these conditions, relaxation of pRYG supercoiled DNA must not be detected.
A major band of 170 kDa was seen when an SDS-PAGE gel was overloaded with the pure protein. There is no 180 kDa topo II form in the final fraction. (Protein concentration is variable from lot to lot but usually is in the range of 5-50 ug per ml.) Top2a may be supplied at 2-10 units/ul (1 unit will decatenate 0.2 ug of KDNA in 30 min at 37°C). Note that unit definitions are based on the preferred DNA substrate, kDNA (kinetoplast DNA). We recommend using kDNA substrates with this enzyme since plasmid DNA relaxation assays are not nearly as sensitive. Activity is certified based on kDNA decatenation (and not on plasmid DNA relaxation).
The final fraction of Human Topoisomerase II Alpha Enzyme comes off an FPLC column in the following buffer: 10% glycerol, 50 mM Tris (pH 7.7), 1 mM EDTA and EGTA, 650 mM NaCl.
