IP-Northern Blot

Homogenize embryo/larval pellet (~200ul) in 2 volumes of lysis buffer (30mM HEPES-KOH pH8/150mM KOAc/5mM Mg(OAC)2/0.1% NP-40 + protease  and RNase inhibitors) in a stainless steel homogenizer. Clear lysate by centrifugation at 10 000xg (S10) followed by centrifugation at 100 000xg (S100).  Incubate with antibody-coupled beads (eg. ANTI-FLAG M2 (Sigma), GFP-Trap (Chromotek), or pre-incubated antibody and protein A/G beads) for 1h at 4°C. Ratio of antibody-coupled beads to amount of protein extract must be optimized on a case-by-case basis. Wash the beads 3x in lysis buffer, and then separate fractions of the beads for protein and RNA analysis. For proteins, resuspend in 2 volumes of 2X SDS-PAGE buffer and elute by heating at 95oC for 5 minutes. Load protein samples onto SDS-PAGE and analyze for your protein of interest. For RNAs, digest beads with proteinase K for 10min at room temp, followed by phenol chloroform extraction of the immunoprecipitated RNA. Precipitate RNA, wash with 70% EtOH, and resuspend the pellet in RNA gel loading buffer II (Ambion). Proceed with northern blot for small RNAs.

Source: Sawh & Duchaine Cell Reports 2013

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