Fluorescence-Based Assays for Microtubule Architecture - Methods in Cell Biology - page 338

29. His60 Ni Resin (Clontech, #635660)
30. Strep-tactin sepharose resin (IBA, Berlin, #2-1201-010)
31. Buffer A (50 mM Na 2 HPO 4 , 300 mM NaCl, pH 7.8)
32. Buffer B (50 mM Na 2 HPO 4 , 300 mM NaCl, 250 mM imidazole, pH 7.8)
33. Strep-tactin wash buffer (100 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl,
pH 8.0)
34. Strep-tactin elution buffer 100 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl,
2.5 mM D-Desthiobiotin (DTB, IBA Berlin, #2-1000-002, 10% glycerol,
pH 8.0)
35. Emulsiflex C3 (Avestin)
36. Disposable plastic columns (Thermo Scientific, # 29922)

21.2 METHODS
21.2.1 Tubulin and microtubule preparations
Tubulin was purified from juvenile bovine brain homogenates by three cycles of po-
lymerization/depolymerization, followed by passage through a phosphocellulose
column, as described in Gell et al. (2011) . Labeling of cycled tubulin with Alexa
Fluor 546 or TAMRA should be performed as described in Hyman et al. (1991) .
Other fluorophores can be chosen to suit the microscope equipment available.

21.2.2 Setup for the single-molecule fluorescence assay
The basics of the single-molecule fluorescence assay are well described in other
chapters in this series, especially in Gell et al. (2011) . In brief, we construct flow
chambers using two silanized cover glass separated by double-stick tape. Microtu-
bules are adhered to the cover glass surfaces by antibodies. The flow chambers allow
us to visualize the surface-immobilized microtubules and exchange solutions in the
chamber during microscopy.

21.2.3 Comparison of controlled 13-pf with controlled 14-pf
microtubules
Two techniques can be used to generate microtubules of controlled thickness:
(1) 13-pf microtubules are nucleated from sea urchin sperm axonemes and (2) 14-pf
microtubules are nucleated using the slowly hydrolyzable GTP analog guanylyl
5 0 - a , b -methylenediphosphonate (GMPCPP). Microtubules nucleated from axonemes
are templated by the A-tubule and central pair of the axoneme and are > 90% 13-pf

( Ray et al., 1993 ). Microtubules nucleated with GMPCPP are > 98% 14-pf ( Meurer-

Grob, Kasparian, & Wade, 2001 ).
Sea urchin sperm axonemes can be purified according to existing protocols based
on sucrose density gradients ( Waterman-Storer, 2001 ). Purified centrosomes can be