Isolation of intraflagellar transport particle proteins from Chlamydomonas reinhardtii

Abstract

Cilia, the hair-like protrusions found on most eukaryotic cells, were once considered vestigial organelles. The recent renaissance of research in cilia arose from the discoveries of intraflagellar transport (IFT) and the involvement of IFT particle proteins in human diseases. Many IFT particle proteins have since been identified, and research on IFT particle complexes and their protein components continues to provide insight into the mechanism of IFT and the etiology of ciliopathies. In this chapter, we describe the methods of isolating IFT particles from the flagella of Chlamydomonas reinhardtii. Two methods, sucrose density gradient fractionation and immunoprecipitation, are explained in detail. Troubleshooting information is presented to illustrate the critical steps of the procedure to ensure successful implementation of these methods in individual labs.