Immunoaffinity purification of DNA polymerase delta.
A monoclonal antibody against human DNA polymerase delta (pol delta) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol delta was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea.
Jiang
Y
Y
Department of Medicine, University of Miami School of Medicine, Florida 33101, USA.
Zhang
S J
SJ
Wu
S M
SM
Lee
M Y
MY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002417:Cattle; D002846:Chromatography, Affinity; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000276:immunology / Q000302:isolation & purification*; D004789:Enzyme Activation; D006801:Humans
D000911:Antibodies, Monoclonal; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase