Labeling Tubulin and Quantifying Labeling Stoichiometry
This is a general procedure for coupling moieties with reactive succinimidyl esters to tubulin. We have used it successfully to derivatize tubulin with succinimidyl esters of biotin, digoxigenin, and a wide range of fluorochromes such as tetramethylrhodamine, X-rhodamine, fluorescein, Oregon Green, Cy3, Cy5 and C2CF (bis-caged carboxyfluorescein). The procedure involves labeling polymeric tubulin, thereby protecting residues important for microtubule assembly. The labeling is performed at high pH to optimize the reaction with the succinimidyl esters and functional tubulin is selected after the labeling reaction by one or more cycles of polymerization and depolymerization.
phosphocellulose-purified tubulin (~50 mg = 2-4 3 ml aliquots of 5-10 mg/ml PC fractions)
Dye stock in anhydrous DMSO (20-100 mM)
BRB80 (1X): 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH (generally made as a 5X stock and stored at 4?C)
High pH Cushion: 0.1 M NaHEPES, pH 8.6, 1 mM MgCl2, 1 mM EGTA, 60% (v/v) glycerol
Labeling Buffer: 0.1M NaHEPES, pH 8.6, 1 mM MgCl2, 1 mM EGTA, 40% (v/v) glycerol
Quench: 2X BRB80, 100 mM K-Glutamate, 40% (v/v) glycerol
Low pH cushion: 60% (v/v) glycerol in 1X BRB80
10X IB (Injection Buffer): 500 mM K-Glutamate, 5 mM MgCl2 (pH of 1X ~ 7.0)
50.2Ti rotor (warm = 37?C)
TLA100.4 or TLA100.3 and TLA100.2 rotors
Small Dounce (2 ml)
Note: 1M HEPES, pH to 8.6 with NaOH and store at -20?C
2M K-Glutamate - dissolve glutamic acid to 2M, carefully pH with KOH such that 50 mM has a pH ~7.0 and store at -20?C
(All buffers for labeling can be stored indefinitely at -20?C; dye stocks are best prepared fresh from powder that has been stored anhydrously at -20?C; residual dye solution can be stored at -20?C or -80?C under anhydrous conditions)