Fig. 4. Measurement of bound and unbound 5xGFP-EMTB in living cells. In order to estimate KD,app, we quantified 5xGFP-EMTB bound to MTs and 5xGFP-EMTB free in the cytoplasm. Using soluble DS-red expressed in the same cells was used as a volume marker. As shown in the figure, images of GFP and DS-red were captured from the same cell. Next, in each image, the background fluorescence was subtracted from an area that contained MTs and regions free of MTs (shown in box). To quantify unbound 5xGFP-EMTB, paired regions that were devoid of microtubules were used to scale the DS-red image to obtain an image of unbound GFP. In the example shown, regions used were 
1x2 µm in size. Bound 5xGFP-EMTB was calculated as (Total GFP) - (Bound GFP). Bound and unbound 5xGFP-EMTB were used to calculate the apparent dissociation constant, KD,app.
