Immunoprecipitation

Homogenize embryo/larval pellet (~200ul) in 2 volumes of 50mM TRIS-HCl pH8/150mM NaCl/1mM EDTA + protease inhibitors in a stainless steel homogenizer. Clear lysate by centrifugation at 10 000xg (S10) followed by centrifugation at 100 000xg (S100). Supplement with Triton X-100 to 1% final concentration. Incubate with primary antibody 1h at 4°C. Ratio of antibody to amount of protein extract must be optimized on a case-by-case basis. Centrifuge at 13 000rpm and add the supernatant to 20-30ul protein A/G beads according to the manufacturer’s instructions. Wash the beads 3x in lysis buffer, resuspend in 2 volumes of 2X SDS-PAGE buffer and elute by heating at 95°C for 5 minutes. Load protein samples onto SDS-PAGE and analyze for your protein of interest.

Source: Sawh & Duchaine Cell Reports 2013

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