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. Author manuscript; available in PMC: 2010 May 15.
Published in final edited form as: Cell. 2009 May 15;137(4):672–684. doi: 10.1016/j.cell.2009.03.035

Figure 5. A Two-Color Method for Locating the Plus Ends of kMTs Relative to the Hec1 Head in Cooled PtK2 Cells.

Figure 5

(A) Fluorescent image of PtK2 cells stably expressing GFP-α-tubulin cooled to 6°C, fixed, and stained with the Hec1 9G3 antibody and a red fluorescent secondary. The image shows a kinetochore fiber and its kinetochore in the same focal plane. At right is a magnified image of the boxed region, showing how linescans were drawn down centers of the fibers through the centroids of Hec1 9G3 fluorescence. (B) Sample linescan of (A) showing GFP-α-tubulin intensity (green) and Hec1 9G3 (red) fluorescent intensity along the linescan. (C) Plot of all normalized linescans (n=92), with Hec1 9G3 centroid set to zero for each on the x-axis and the error function (purple) that best fits the data set. Blue lines mark x and y positions of the 50% amplitude of the error function.