Site-Specific Fluorescent Labeling of Tubulin - Methods in Cell Biology - page 8

7-(Diethylamino) coumarin-3-carbohydrazide : Dissolve the commercially avail-
able CZ in DMSO to a final concentration of 5 mM or higher. Determine the concen-
tration spectrophotometrically using an extinction coefficient of 46,000 M 1 cm 1 at
420 nm. The stock solution can be stored at 20 C. Vortex well before use. Keep in

the dark when not in use.

1.2.4.3 Preparation of fluorophore-labeled tubulin
Prepare a solution of the 3fY ligated tubulin (3fY-Tb) in PME containing 10 mM
4aF. Allow this solution to react with a fluorophore of choice in 1:10 molar ratio
(protein:fluorophore) in the dark, at 4 C for 60 min or at room temperature for
2h( Banerjee, Panosian, et al., 2010; Blanden, et al., 2011 ). It would be prudent
to empirically determine the incubation times for the selected fluorophore the first
time the procedure is performed. Desalt fluorescent protein into PME or a chosen
buffer to remove excess fluorophore and 4aF. This material can be drop-frozen
and stored in liquid nitrogen until use.

1.2.5 Coupling biotin to the C-terminus of -tubulin

a

1.2.5.1 BH stock preparation
Make a 50 mM BH stock in DMSO. Store the stock at 4 C until use. Vortex well
before use.

1.2.5.2 Biotinylation of tubulin
Prepare 3fY-Tb in PME containing 10 mM 4aF. Allow this solution to react with BH
in 1:10 molar ratio (protein: BH) at 4 C for 2 h. Desalt biotinylated protein into PME
or a chosen buffer to remove excess BH and 4aF. This material can be drop-frozen
and stored in liquid nitrogen until use.

1.2.6 Detection of modification in the C-terminus of tubulin
1.2.6.1 Detection of tyrosine or 3-fomyltyrosine in the C-terminus
of a -tubulin
Mix the protein sample with sample buffer and run SDS-PAGE according to the proce-
dure described by Banerjee, Bovenzi, et al. (2010) . Standard protocols often will not
separate the dimer. Equilibrate the gel in transfer buffer for 45 min and then transfer

the protein samples forWesternblot ontoa polyvinylidene fluoridemembrane. Incubate
themembrane inblockingbuffer for 2 h at roomtemperature ona rocker. Thenprobe the
membranewith TUB-1A2 antibody (1:2000) for 90 min at room temperature. Wash off
the excess antibody with PBST and then probe the membrane with goat antimouse sec-
ondary antibody HRP conjugate (1:5000) for 60 min at room temperature. Wash three
times with PBST and develop with the blot with TMB solution ( Fig. 1.1 ).

ab