Dear all, I'm doing RNA pulldown experiments to identify RNA-binding proteins which interact with my RNA. I biotinylated my RNA using biotin RNA labeling mix (roche) and PAGE purified the RNA. For binding, I overnight incubated 5 ug (~80 pmoles) of biotinylated RNA with 1.6 mg of Hela nuclear extract in the presence of  0.1 ug/ul tRNA and 1U/ul RNasin. For pulldown, I added 30 ul of streptavidin agarose beads (Thermo) to the binding reaction and incubated for 1 hr. After extensitve washing, I eluted captured proteins in SDS sample buffer and performed SDS-PAGE. Following coomassie staining, I could observe several bands specifically enriched in the RNA(+) sample, but the problem is that there are too many non-specific proteins appearing in both beads-only and RNA(+) samples to precisely excise out bands of interest without contamination. I tried pre-clearing my extract  by incubating it with 60 ul of streptavidin beads before binding, but this did not significantly improve specificity. Can anybody help me reduce non-specific protein binding on beads? Any comments would be greatly appreciated. 

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