Mitchell Kimberly A P
Department of Biology, Liberty University, Lynchburg, Virginia.
Curr Protoc Cell Biol. 2013 Jun;Chapter 3:3.42.1-3.42.9. doi: 10.1002/0471143030.cb0342s59.
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-µm long in Xenopus A6 toad kidney cells as opposed to 1 to 5 µm in NIH3T3 fibroblast cells).
细胞的初级纤毛既是一种机械传感器，也是一种参与多种信号通路的化学传感器。为了确定初级纤毛中的蛋白质富集情况或研究各种蛋白质在纤毛下的定位，将初级纤毛从细胞体中分离出来是很有必要的。本文所述的方案提供了通过剪切力将初级纤毛与细胞体分离来纯化初级纤毛的详细说明。这种简单的技术避免了使用可能影响纤毛中信号蛋白或导致纤毛膜解体的苛刻纯化条件。此外，由于细胞体大多保持完整，细胞体中的蛋白质对分离出的纤毛的污染降至最低。该方案非常适合从肾细胞系中分离纤毛，因为这些细胞中的初级纤毛比其他细胞类型中的长得更长（非洲爪蟾A6蟾蜍肾细胞中长达50微米，而NIH3T3成纤维细胞中为1至5微米）。
