Site-Specific Fluorescent Labeling of Tubulin - Methods in Cell Biology - page 4

Fortunately, it is possible to perform the coupling reaction at higher pH by using an
aromatic amine as a catalyst. Aniline at a final concentration of 100 mM is the most
common catalyst ( Dirksen, et al., 2006 ). We do not use aniline for catalyzing reac-
tions with tubulin, however, as it also serves as a denaturant at concentrations nec-
essary for efficient catalysis. We have found that the commercially available
4-aminophenylalanine satisfactorily catalyzes the coupling reaction at neutral pH
( Blanden, Mukherjee, Dilek, Loew, & Bane, 2011 ). Furthermore, the catalyzed
reaction can be performed efficiently at low temperature (4 C), which further
protects tubulin from denaturation during the labeling process.
The protocol presented here describes tubulin labeling with fluorophores. The
method likely to be amenable to other applications (such as attaching tubulin to a
bead, a surface, a nanoparticle, etc.) provided that the reacting partner with an
appropriate functional group can be obtained. One such example, biotinylation of
tubulin, is described.

1.1 MATERIAL
1.1.1 TLL expression and purification
1. EX-T8583-B05 (Genecopoeia, MD)
2. Luria Bertani (LB) broth and LB agar (Sigma-Aldrich, MO)
3. Ampicillin (Sigma-Aldrich)
4. Glutathione Sepharose 4B resin (GE Healthcare, Sweden)
5. 1 M Isopropyl - D -1-thiogalactopyranoside (IPTG) (Gold Biotechnology, MO)

solution in water
6. TE buffer (10 mM Tris, 1 mM EDTA, pH 8) containing 0.3 mg of PMSF
(phenylmethanesulfonyl fluoride) per mL
7. PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4)
8. Elution buffer (50 mM Tris, 20 mM reduced glutathione, pH 8.0)

b

1.1.2 Labeling tubulin
1. PME buffer (0.1 M PIPES, 1 mM MgSO 4 , 2 mM EGTA, pH 6.90)
2. Sephadex G50 and G75 resin (GE Healthcare)
3. CPA from bovine pancreas (Type II-PMSF treated), 50 units mg 1 protein, an

aqueous suspension (Sigma-Aldrich, Catalog # C9268)
4. TTL buffer (25 mM MES, 150 mM KCl, 27

m M MgCl 2 , 2.5 mM ATP, 1 mM

DTT, 1.5% glycerol, pH 6.8)
5. 3fY
Note : This unnatural amino acid is presently not available commercially. It can be
prepared by a two-step synthesis process from commercially available starting
material (BOC- L -tyrosine) and common laboratory chemicals ( Banerjee, Panosian,
et al., 2010 )
6. EZ-linked biotin hydrazide (BH) (Thermo Scientific, IL)