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

Both samples were precipitated in methanol:chloroform (4:1) and resuspended in 0. LoBind microcentifuge tubes (Eppendorf, Hamburg, Germany) and centrifuged at 10,000 g for 682 10 minutes at 4°C to pellet cilia. The pellet was gently resuspended and diluted to 600µl with 683 33% artificial seawater containing 100mM DTT. The cilia sample was overlain onto a sucrose 684 step gradient from 80-30% sucrose (sucrose solution contained 50mM Tris, pH8.0, 10mM DTT), 685 step size 10%, 1ml per step. The sample was spun at 100,000 g in a swinging bucket rotor for 3 686 hours at 4°C and fractions were collected from the bottom of the tube by puncturing with a 30 687 gauge needle and allowing the sample to flow by gravity. A portion of each fraction was 688 analyzed by immunoblot to determine which fractions contained high levels of TUB ac and low 689 levels of Actin ( Figure 1C , e.g. fractions 6 and 7). To concentrate the samples and remove the 690 sucrose, the protein was precipitated using 16% trichloroacetic acid (TCA) and washed 2 times 691 with acetone, then resuspended in RapiGest SF Surfactant for mass spectrometry. 692