Isolation and Analysis of Flagella Proteins

To isolate flagella for protein analysis, we followed a protocol described by Witman (1986) (link). In brief, cells were concentrated and washed in 10 mM HEPES, pH 7.4, resuspended in HMS at 4°C (10 mM HEPES, 5 mM MgSO₄, and 4% sucrose) and deflagellated by adding dibucaine to a final concentration of 4.17 mM (Sigma-Aldrich) and repeated pipetting. Flagella were separated from cell bodies by centrifugation, collected from the supernatant by centrifugation, and resuspended in HMEK (30 mM HEPES, 5 mM MgSO₄, 25 mM KCl, and 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA]) with protease inhibitor cocktail (Sigma-Aldrich). For Western blotting, SDS–PAGE sample buffer was added to flagella samples and samples were incubated at 85°C for 10 min. The following primary antibodies were used: mouse anti-IC2 (1:1000; King and Witman, 1990 (link)), mouse anti-IFT81 (1:1000; Cole et al., 1998 (link)), rabbit anti-ODA16 (1:200; Ahmed and Mitchell, 2005 (link)), and mouse anti-NG (1:1500; Chromotek). Western blots were developed using anti-mouse and anti-rabbit immunoglobulins G conjugated to horseradish peroxidase (Invitrogen) and chemilu­minescence substrate (Michigan Diagnostics). Images were captured using a BioRad Gel Doc imaging system.