Isolation of Primary Cilia by Shear Force

Author(s)

Kimberly A. P. Mitchell, Liberty UniversityFollow

Publication Date

6-2013

Document Type

Article

Disciplines

Biology | Microbiology

Comments

This article was published in Current Protocols in Cell Biology: http://onlinelibrary.wiley.com/book/10.1002/0471143030.

Abstract

The cell’s primary cilium is both a mechanical and chemical sensor involved in many signaling pathways. In order to ascertain protein enrichment in the primary cilium or study sub-ciliary localization of various proteins, it is advantageous to remove the primary cilium from the cell body. The protocol described here gives detailed instructions on purifying primary cilia by separating them from the cell body using shear force. This simple technique avoids using harsh purification conditions that may affect signaling proteins in the cilium or cause the ciliary membrane to disintegrate. In addition, as the cell body remains mostly intact, contamination of the isolated cilia by proteins from the cell body is minimized. This protocol is ideally suited for isolating cilia from renal cell lines, as primary cilia in these cells grow to greater lengths than in other cell types (up to 50 mm long in Xenopus A6 toad kidney cells as opposed to 1-5 mm in NIH3T3 fibroblast cells).

Recommended Citation

Mitchell, Kimberly A. P. (June 2013). "Isolation of Primary Cilia by Shear Force." Current Protocols in Cell Biology. http://www.currentprotocols.com/WileyCDA/CPUnit/refId-cb0342.html.