2′-OMe pull-down

Homogenize embryo/larval pellet (~200ul) in 2 volumes of  lysis buffer (25 mM Hepes-KOH pH7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 % glycerol, 0.5 % Triton X-100 and protease inhibitors) using a stainless steel homogenizer. Pre-clear S10 lysate with 25uL of m-280 streptavidin beads (Invitrogen) and an unrelated 2’O-methylated oligonucleotide (0.1mmol anti-miR-35) for 1h at 4oC with rotation. Incubate the supernatant  with biotinylated 2’-O-Me oligonucleotides (0.1mmol) complementary to your miRNA of interest (or human miR-16 as a negative control) for 1 hour at 22oC. After centrifugation for 5min at 13000rpm, incubate the supernatant with 25mL of m-280 streptadividin beads for 30 minutes at 4oC. Wash beads three times using ice-cold lysis buffer containing 0.1% Triton X-100 and 2mM DTT, followed by a wash without detergent and 2mM DTT. Resuspend beads in 50uL of 2X SDS loading buffer and elute by heating at 95oC for 5 minutes. Load protein samples onto SDS-PAGE and analyze for your protein of interest.

Source: Sawh & Duchaine Cell Reports 2013