FLAG IP

IPs with FLAG-tagged protein were performed with Protein G magnetic beads (Invitrogen™ Dynabeads™ Protein G; #10004D) and α-FLAG antibody (Monoclonal ANTI-FLAG® M2 antibody produced in mouse, Sigma Aldrich, #F3165). Per IP, 30 µl of beads were used and washed three times with 1 ml Wash Buffer (25 mM Tris/HCl pH 7.5, 300 mM NaCl, 1.5 mM MgCl₂, 1 mM DTT, 1 complete Mini protease inhibitor tablet per 50 ml). The beads were resuspended in 450 µl Wash Buffer and up to 1 mg of complete protein extract from the respective C. elegans strains was added. Finally, 2 µg of FLAG antibody were added and the samples were incubated for 3 h, rotating at 4 °C. After the incubation, the samples were washed three to five times with 1 ml Wash Buffer (see washing steps before), the beads were resuspended in 1x LDS/DTT, and the samples were boiled at 95 °C for 10 min. For mass spectrometry, IPs were prepared in quadruplicates per strain/condition. When doing the IP with Sm nuclease, the wash buffer was supplemented with 0.05% Sm nuclease (as indicated above).