Biology Reference
In-Depth Information
FIGURE 1.1
Detection of tyrosine or 3-fomyltyrosine in the C-terminus of
-tubulin: Detyrosinated
tubulin was incubated with tubulin tyrosine ligase in the presence of either
L
-tyrosine
or 3-formyltyrosine as described and then subjected to SDS-PAGE. Top: Western blot
with tyrosinated a-tubulin antibody (Sigma-Aldrich TUB-1A2). Bottom: Coomassie stain.
The first protein band in the gel is
a
a
-tubulin and the second band is
b
-tubulin. The
mass of protein loaded into each well was 1
m
g for the Western blot and 10
m
g for the protein
stain.
1.2.6.2
Detection of fluorophore-labeled tubulin
Separate
ab
dimer by SDS-PAGE and observe
a
-tubulin in a dark room using long-
wavelength UV lamp (
Fig. 1.2
).
The ratio of the fluorophore to tubulin dimer may be estimated by UV spectros-
copy and protein assay. From the UV spectrum of the final product, calculate
the concentration of the fluorophore using extinction coefficients listed above.
Determine the protein concentration by protein assay (e.g., BCA). The overall
labeling efficiency is typically up to 0.3 mol of fluorophore per mol of tubulin.
1.2.6.3
Detection of biotinylated tubulin
To detect biotinylated tubulin, perform a Western blot as described previously and
probe the blot with streptavidin-HRP conjugate (1:10,000) (
Fig. 1.3
).
1.2.7
Evaluation of tubulin assembly activity
Equilibrate protein samples (5
M final concentration) in PME buffer at 37
C
and record the absorbance at 350 nm to obtain the baseline. Add 5
m
M taxol in
DMSO (final concentration 2% v/v) to initiate polymerization. Detect the extent
of polymerization by monitoring apparent absorption at 350 nm over
m
time
(
Fig. 1.4
).
