Evolutionary proteomics uncovers ciliary signaling components

Monika Abedin Sigg; Tabea Menchen; Jeffery Johnson; Chanjae Lee; Semil P. Choksi; Galo Garcia; Henriette Busengdal; Gerard Dougherty; Petra Pennekamp; Claudius Werner; Fabian Rentzsch; Nevan Krogan; John B. Wallingford; Heymut Omran; Jeremy F. Reiter

To separate the flagella from the cell bodies, the sample was split in half and purified 711 over Percoll as described above. The top, clear layer containing the flagella was collected and the 712 pellet and bottom 2 ml containing most of the cells were discarded. The flagellar fraction was 713 spun at 16,000 g for 20 minutes at 4°C to pellet the flagella. To further purify the sample, the 714 flagellar pellet was gently resuspended in ~800µl of artificial seawater and overlain onto a 715 sucrose step gradient. The sucrose was dissolved in 10mM Hepes, pH7.4, 5mM MgSO 4 , 5% 716 artificial seawater and a step gradient from 40-80% sucrose was prepared using 10% steps, 1ml 717 of each step. The sample was spun at 100,000 g for 3 hours at 4°C. To collect fractions, a hole 718 was punctured in the bottom of the tube using a 25 gauge needle and fractions between 100 and 719 700µl were captured. 720 A small portion of each fraction was analyzed by immunoblot to determine which 721 fractions contained high levels of TUB ac and low levels of Actin ( Figure 1G, Fractions 4 and 5) . 722