Site-Specific Fluorescent Labeling of Tubulin - Methods in Cell Biology - page 7

1.2.3.1.2 3-Formyltyrosine
Prepare a 30 mM solution of 3fY in PME buffer. This concentration is near
the limit of the molecule's solubility, and dissolution of the solid requires repeated
vortexing and sonication. Centrifuge the solution at 5600 g for 10 min to remove

any undissolved solute. Determine the actual concentration of the clarified 3fY
stock spectrophotometrically using an extinction coefficient of 10,715 M 1 cm 1
at 257 nm. Store the solution at 4 C until use. (This solution can be stored at
4 C for months.) Equilibrate the solution to room temperature and vortex well be-
fore use.

1.2.3.2 Coupling reaction
Allow the detyrosinated tubulin from section 1.2.2 in TTL buffer to react with excess
ligand (Y or 3fY) in the presence of GST-TTL (final concentrations: 0.2 mg per mL
GST-TTL, 1 mM Y or 2 mM 3fY) for 20-30 min at 37 C. Next, remove the excess
ligand by rapid gel filtration into PME buffer using G-50 resin. Note that the elutant
will contain both 3-formyltyrosinated tubulin (3fY-Tb) and GST-TTL. This material
can be drop-frozen in liquid nitrogen and stored in liquid nitrogen until use.
The presence of GST-TTL in the tubulin solution at this ratio does not affect
taxol-induced tubulin assembly, and no further processing of the protein mixture
is performed for experiments in which the presence of GST-TTL is unimportant.
To obtain pure tubulin, remove GST-TTL using glutathione Sepharose 4B in
PME. We find it convenient to add the appropriate volume of resin to the reaction
mixture before removing the excess ligand through rapid gel filtration; thus, this step
removes both enzyme and ligand together.

1.2.4 Fluorescent labeling of tubulin
1.2.4.1 Preparation of catalyst stock
Prepare 50 mM stock solution of 4aF in PME, pH 6.9. Repeated sonication and vor-
texing is required to dissolve all 4aF at this concentration. Store the stock at 4 C until
use. Warm the solution to room temperature and vortex well before use.

1.2.4.2 Preparation of fluorophore stock
Texas Red hydrazide : Dissolve Texas Red hydrazide in DMSO to a final
concentration of 5 mM or higher. Determine the concentration spectrophotometri-
cally using an extinction coefficient of 109,000 M 1 cm 1 at 588 nm. The stock
solution can be stored at

20 C. Vortex well before use. Keep in the dark when

not in use.
CH : Just before use, prepare CH in PME to a final concentration of 500

m

Mor
higher. Dissolve CH in PME by repeated vortexing and sonication. Centrifuge at
5600

g for 10 min to remove any undissolved solute. Check the concentration
of the solution after centrifugation by UV-Vis spectroscopy using extinction coef-
ficient of 19,000 M 1 cm 1 at 346 nm. Keep in the dark when not in use.