200,000 cpms of a radioactively body-labeled RNA were washed in 70% ethanol, dried and resuspended in 16 µL water. To this solution, 10 µg of yeast tRNAs and 20 µL of a nuclear extract prepared from HeLa cells (~30 µg/µL)were added. This mixture was incubated for 15 min on ice, exposed to UV light for 15 min on ice (UV Stratalinker™ 2400, lambda=254 nm, Stratagene), and treated with Rnase A at a 1 µg/µL final concentration for 15 min at room temperature. Meanwhile, 20 µL of a 50% suspension of Dynabeads coupled to protein A (Invitrogen) were washed once with 1 mL PBS and pre-blocked 30 min at room temperature with 0.2% BSA complemented PBS buffer under gentle rotation (16 rpms). The resin was washed three times with 1 mL of NET-2 buffer (50mM Tris.HCl pH 7.9, 100 mM NaCl, 0.05% NP40, 0.5 mM DTT) and pre-blocked again for 1 hr at 4°C in 5% BSA complemented PBS. Following three successive washes in NET-2 buffer, the beads were resuspended in a final volume of 42.5 µL of NET-2 buffer and incubated for 1 hr at room temperature with 3 µg of antibody, under gentle rotation. Antibody-coated beads were washed three times in 1 mL of NET-2 buffer and incubated for 1 hr at 4°C with to the former mixture of nuclear extract with radioactive RNA and tRNA, under gentle rotation. The unbound proteins were washed from the beads three times with 1 mL of NET2 buffer and elution occurred by addition of 12 µL of 1X sample solution and heating 5 min at 95°C.
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