Labeling Tubulin and Quantifying Labeling Stoichiometry

• I. Solutions & Supplies
  
  
• II. Labeling Protocol
  
  
• III. Quantifying Tubulin Concentration and Labeling Stoichiometry
  
  
• IV. Using Labeled Tubulins
  
  
• V. Properties of Fluorescent Dyes Used for Tubulin Labeling
  

Back to protocols

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 MgCl_2, 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)

1. Thaw 2-4 PC column fractions (30-60 mg tubulin) and add BRB80 to 0.5X, MgCl₂ to 3.5 mM , GTP to 1 mM and store on ice for 5'. Transfer to 37¡C and add DMSO to 10% final in two steps, mixing gently but thoroughly and incubate at 37¡C for 30 min. Alternatively, add half volume of glycerol to promote polymerization. In a side-by-side comparison, for reasons that are not clear, using DMSO instead of glycerol for the first polymerization step appears to increase the labeling stoichiometry by ~25% for C2CF-SNHS ester. For labeling with rhodamine (tetramethylrhodamine NHS ester) and X-rhodamine, we generally use glycerol polymerization.

2. Layer polymerized tubulin onto 20 ml warm High pH Cushion in two 50.2 Ti tubes. Pellet microtubules in a Beckman ultracentrifuge in a 50.2 Ti rotor at 40K for 45' at 35¡C.

3. Aspirate the supernatant above the cushion and rinse the supernatant-cushion interface twice with warm (37¡C) Labeling Buffer . Aspirate the cushion and resuspend the pellet using a cutoff large pipet tip in 1 - 2 ml of warm labeling buffer. Take care to keep the tubulin warm during the resuspension and continue resuspending till no chunks of tubulin are visible. This is the most painful part of the labeling procedure.

4. Add 10- to 20-fold molar excess of the dye to tubulin. Estimate the tubulin concentration assuming ~70% recovery of the starting tubulin. For dyes such as Cy5 and Cy3, use a 5-pack for labeling ~25 mg. For most dyes we label for 30'-40' at 37¡C. For C2CF-SNHS (caged fluorescein), we have found it best to add the dye in two steps (20' apart) and label for 60' at 37¡C. After adding the dye stock, gently vortex the mixture every 2'-3' during the course of the labeling.

5. At end of labeling incubation add an equal volume of Quench to the labeling reaction and mix well. Incubate for 5'.

6. Layer the quenched labeling reaction onto two TLA100.3 (or TLA100.4) tubes containing 1.5 ml of Low pH Cushion . Spin at 80K for 20 min at 35¡C in a TLA100.3 or TLA100.4 rotor in a Beckman TLA100 ultracentrifuge.

7. Aspirate the supernatant above the cushion and rinse the supernatant-cushion interface twice with warm 1XBRB80. Aspirate the cushion and resuspend the pellet using a cutoff pipet tip in 1 ml of ice-cold 1X IB . Transfer resuspended chunks of the pellet to a small ice-cold dounce (1 or 2 ml volume) in an ice-water bath. Resuspend the pellet by gentle douncing till the suspension is uniform. Continue douncing intermittently for a total time of 30 min at 0¡C.

Cold IB seems to promote more rapid depolymerization than BRB80; therefore, we use IB in the depolymerization step for all labeling procedures. For small scale labelings the pellet can be resuspended directly in the centrifuge tube and sonicated gently using a microtip sonicator to speed depolymerization.

8. Spin the depolymerized tubulin in a TLA100.2 (or TLA100.3) rotor at 80K for 10' at 2¡C.

9. Recover the supernatant from the cold spin, add BRB80 to 1X (from a 5X stock), MgCl₂ to 4 mM, GTP to 1 mM and incubate on ice for 3'. Warm to 37¡C for 2', add 1/2 volume of glycerol (33% v/v final), mix well and polymerize at 37¡C for 30 min.

10. Layer the polymerization reaction on a 1 ml Low pH Cushion in a TLA100.3 tube and pellet the microtubules at 80K in a TLA100.3 rotor for 20' at 37¡C.

11. Aspirate the supernatant above the cushion and rinse the supernatant-cushion interface twice with warm IB . Aspirate the cushion and rinse the pellet twice with 1 ml warm IB to remove any residual glycerol. Resuspend the pellet using a cutoff pipet tip in 0.2-0.3 ml of ice cold IB . This pellet should resuspend easily. Incubate at 0¡C for 20 to 30 min.

12. Spin the depolymerized tubulin in a TLA100 or TLA100.2 rotor at 80K for 10' at 2¡C. Recover the supernatant, quickly estimate the tubulin concentration, adjust with IB if desired and freeze in 3 - 5 ul aliquots in liquid nitrogen. We generally aim for a final tubulin concentration of 5 - 15 mg/ml (50 - 150 µM). Careful determination of tubulin concentration and labeling stoichiometry can be performed as described below, after the tubulin has been aliquoted and frozen. C2CF-tubulin should be stored at -80¡C in a foil-wrapped box.

An example of calculating concentration and stoichiometry for tubulin labeled with tetramethylrhodamine (TMR) NHS ester:

Tubulin concentration = [(A₂₈₀ - Contribution of dye to A₂₈₀ ) x Dilution Factor] / Extinction

coefficient of tubulin at 280 nm

TMR concentration = (A₅₅₅ x Dilution Factor) / Extinction Coefficient of TMR at 555 nm

Labeling Stoichiometry = TMR concentration / Tubulin concentration

A wavelength spectrum of 1/100 dilution of the final labeled tubulin product gave the following absorbance values:

A₂₈₀ = 0.23; A₅₅₅ = 0.20

Therefore, Tubulin concentration = [{0.23 - (0.2 x 0.2)} x 100]/115000 = 165 µM

TMR concentration = [0.20 x 100]/95000 = 210 µM

Labeling Stoichiometry = 210/165 = 1.3

To determine the concentration and labeling stoichiometry of C2CF-tubulin, the C2CF must be first uncaged to fluorescein. To do this, dilute the labeled tubulin 1/50 to 1/100 in IB + 2 mM DTT in an eppendorf tube. Put the eppendorf tube on a hand held UV lamp, cover with foil (shiny side down) and expose to long wavelength UV for 30'. Obtain a wavelength spectrum from 200 to 600 nm after the 30' activation, using IB + 2 mM DTT exposed to UV in parallel as a blank. Assuming a 100% efficiency for the uncaging reaction, the concentration of C2CF can be calculated from the spectrum after activation as follows:

Concentration of C2CF = (A₄₉₅ x Dilution Factor x 1.2)/74000

(The factor of 1.2 corrects for the pH dependence of the absorption spectrum of fluorescein)

e _max = Extinction coefficient of dye at its peak wavelength

e ₂₈₀ /e _max = Absorbance of dye at 280 nm as a fraction of its absorbance at its peak wavelength

5 (and-6) carboxyfluorescein succinimidyl ester ( Molecular Probes C-1311)

Oregon Green 488 carboxylic acid, succinimidyl ester 5-isomer ( Molecular Probes O-6147)

5 (and-6) carboxytetramethylrhodamine succinimidyl ester ( Molecular Probes C-1171)

5 (and-6) carboxy-X-rhodamine succinimidyl ester ( Molecular Probes C-1309)

Texas Red-X succinimidyl ester, mixed isomers ( Molecular Probes T-6134)

Cy3-OSu monofunctional reactive fluorophore ( Amersham PA13100)

Cy5-OSu monofunctional reactive fluorophore ( Amersham PA13600)