cDNA corresponding to the first 100 amino acids of OEF-1 was cloned into the pET-19b expression vector (Novagen) and transformed into BL21 cells. His-tagged OEF-1 N-terminal fragments were purified over Nickle-NTA resin (Qiagen) under denaturing conditions, and injected into rabbits (Pocono Rabbit Farm and Laboratory). Affinity purification of the bleeds was performed as in PORTER AND KOELLE 2010. Briefly, purified OEF-1 N-terminal protein was run on an acrylamide gel and transferred onto nitrocellulose. The bleeds were incubated with the membrane overnight at 4 C, and bound antibodies were eluted off the membrane with 100 mM glycine (pH 2.5).
oef-1