Generation of Anti-DAZL1 Antibody
A 229-base pair Sau3AI fragment encoding the last 76 amino acid residues of DAZL1 was isolated from a human DAZL1 cDNA clone f2 and cloned into the BamHI site of an expression vector pET32b (Novagen, Madison, WI). The construct was introduced into Escherichia coli to direct the synthesis of a fusion protein between thioredoxin and DAZL1, which was purified on His-Bind metal chelation resins according to the manufacturer’s manual, and injected into rabbits to generate antibody.
In Vitro RNA Binding Assay
S35-Labeled proteins were synthesized in vitro using the TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI). In vitro binding of the labeled proteins to RNA homopolymers was carried out according to Siomi et al.. Briefly, equal amounts of S35-labeled proteins were added to 250 ^l of a 10% suspension of RNA homopolymers immobilized on agarose beads in a binding buffer containing 10 mM Tris pH 7.4, 2.5 mM MgCl2, 0.5% Triton X-100, 2 mg/ml heparin, and 0.1 M NaCl unless otherwise stated. After rocking at 4°C for 10 min, the beads were washed 5 times with 1 ml of ice-cold binding buffer. Bound proteins were eluted by heating in 50 ^l of double-strength Laemmli sample buffer at 100°C for 5 min. After cooling on ice immediately afterward, the beads were spun down, and analysis of the supernatants on 10% SDS-PAGE gels was followed by autoradiography. Poly U, poly C, and poly G were purchased from Sigma (St. Louis, MO), and poly A was purchased from Pharmacia (Piscataway, NJ).