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MOLECULAR BIOLOGY: WORKING WITH DNA

PROTEIN EXPRESSION: LABELING

Labeling Tubulin with Fluorescent Dye and Quantifying Labeling Stoichiometry

Labeling Tubulin with Fluorescent Dye and Quantifying Labeling Stoichiometry
Contributor: The Laboratory of Timothy Mitchison at Harvard University
 
Overview
This is a general procedure for coupling moieties with reactive succinimidyl esters to tubulin. This protocol has been used 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).
 
Procedure
A. Labeling Tubulin (See Hint #1 and Hint #3).

1. Thaw 30 to 60 mg tubulin and add BRB80 to a final concentration of 0.5X, MgCl2 to 3.5 mM, and GTP to a final concentration of 1 mM.

2. Incubate on ice for 5 min.

3. Transfer to 37°C bath and add DMSO to a final concentration of 10% (v/v) and mix gently but thoroughly (See Hint #4).

4. Incubate at 37°C for 30 minutes.

5. Place 20 ml of High pH Cushion into two ultracentrifuge tubes.

6. Layer polymerized tubulin onto the High pH Cushion.

7. Centrifuge in an ultracentrifuge at 145,500 X g (40,000 rpm using a Type 50.2Ti rotor) for 45 min at 35°C.

8. Aspirate the supernatant without removing the cushion or the pelleted tubulin.

9. Rinse the cushion supernatant interface two times using pre-warmed (37°C) Labeling Buffer.

10. Aspirate the cushion.

11. Resuspend the pellet using a cutoff large pipette tip in 1 to 2 ml of pre-warmed (37°C) Labeling Buffer (See Hint #5).

12. Add 10 to 20-fold molar excess of the dye in Dye Stock to the tubulin (See Hint #6).

13. Incubate for 30 to 40 minutes at 37°C. After adding the dye stock, gently vortex the mixture every 2 to 3 min during the course of the labeling (see Hint #7).

14. To end the labeling, add an equal volume of Stop Solution and mix well.

15. Incubate in Stop Solution for 5 min.

16. Place 1.5 ml of Low pH Cushion into two ultracentrifuge tubes.

17. Layer labeled tubulin onto the Low pH Cushion.

18. Centrifuge in an ultracentrifuge at 264,500 X g (80,000 rpm using a TLA100.3 rotor) for 20 min at 35°C.

19. Aspirate the supernatant without removing the cushion or the pelleted tubulin.

20. Rinse the cushion-supernatant interface two times using pre-warmed (37°C) 1X BRB80.

21. Aspirate the cushion.

22. Resuspend the pellet using a cutoff large pipette tip in 1 ml of ice-cold Injection Buffer (See Hint #8).

23. Transfer labeled tubulin to a small ice-cold dounce. Keep dounce in an ice-water bath.

24. Gently dounce the sample until the suspension is uniform.

25. Continue douncing intermittently for a total time of 30 min at 0°C to promote depolymerization of the microtubules.

26. Centrifuge the depolymerized tubulin in ultracentrifuge at 264,500 X g (80,000 rpm using a TLA100.3 rotor) for 10 min at 2°C.

27. Recover the supernatant and add BRB80 to a final concentration of 1X, MgCl2 to 4 mM, and GTP to a final concentration of 1 mM.

28. Incubate on ice for 3 min.

29. Incubate at 37°C for 2 min.

30. Add an one-half volume of 100% Glycerol (33% (v/v) final concentration) and mix well.

31. Incubate to polymerize tubulin for 30 min at 37°C.

32. Place 1 ml of Low pH Cushion to into two ultracentrifuge tubes.

33. Layer labeled tubulin onto the Low pH Cushion.

34. Centrifuge in ultracentrifuge at 264,500 X g (80,000 rpm using a TLA100.3 rotor) for 20 min at 37°C.

35. Aspirate the supernatant without removing the cushion or the pelleted tubulin.

36. Rinse the cushion-supernatant interface two times using 1 ml of pre-warmed (37°C) Injection Buffer.

37. Aspirate the cushion.

38. Rinse the pellet twice with 1 ml of pre-warmed (37°C) Injection Buffer to remove any residual Glycerol.

39. Resuspend the pellet using a cutoff large pipette tip in 200 to 300 μl of pre-warmed (37°C) Injection Buffer (see Hint #9).

40. Incubate in an ice-bath for 20 to 30 min.

41. Centrifuge in an ultracentrifuge at 264,500 X g (80,000 rpm using a TLA100.2 rotor) for 10 min at 2°C.

42. Recover the supernatant. Quickly estimate the tubulin concentration, adjust the concentration of tubulin to between 5 to 15 mg/ml (50 to 150 μM) by adding ice-cold Injection Buffer (See Hint #10).

43. Aliquot labeled tubulin and store at -80°C (See Hint #11).

B. Quantifying Tubulin Concentration and Labeling Stoichiometry

1. Dilute the labeled tubulin from one-fiftieth to one-one hundredth in Injection Buffer.

2. Using the diluted tubulin samples, obtain a wavelength spectrum.

3. Calculate the molar concentration of dye by using the absorbance at the peak wavelength and the extinction coefficient provided by the dye manufacturer.

4. Determine the absorbance at 280 nanometers (Abs280) of the labeled tubulin.

5. Subtract from the Abs280 value of the labeled tubulin from the absorbance 280 of the dye.

6. Calculate the protein concentration of the tubulin using an extinction coefficient of 115,000 M1 cm1 (See Hint #12).

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

Tubulin concentration = [(Abs280 - Contribution of dye to Abs280) x Dilution Factor]/Extinction coefficient of tubulin at 280 nm.

TMR concentration = (Abs555 x Dilution Factor)/Extinction Coefficient of TMR at 555 nm

Labeling Stoichiometry = TMR concentration/Tubulin concentration

Extinction Coefficient of TMR is 95000 M1 cm1

A wavelength spectrum of 1/100 dilution of the final labeled tubulin product gave the following absorbance values: Abs280 = 0.23; Abs555 = 0.20

Therefore, Tubulin concentration = [{0.23 - (0.2 x 0.2)} x 100]/115,000 = 165 μM

TMR concentration = [0.20 x 100]/95,000 = 210 **M

Labeling Stoichiometry = 210/165 = 1.3

8. To determine the concentration and labeling stoichiometry of C2CF-tubulin, the C2CF must be first uncaged to fluorescein.

a. To do this, dilute the labeled tubulin from one-fiftieth to one-one hundredth in Injection Buffer containing 2 mM DTT in a microcentrifuge tube.

b. Put the microcentrifuge tube on a hand held UV lamp, cover with foil (shiny side down).

c. Expose to long wavelength UV for 30 min.

d. Obtain a wavelength spectrum from 200 to 600 nm after the 30 min activation, using Injection Buffer containing 2 mM DTT exposed to UV in parallel as a blank.

e. Assuming a 100% efficiency for the uncaging reaction, and that the extinction coefficient of C2CF is 74,000 M1 cm1, the concentration of C2CF can be calculated from the spectrum after activation as follows:

Concentration of C2CF = (Abs495 x Dilution Factor x 1.2)/74,000

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

C. Using Labeled Tubulin for Microinjections into Cells

1. Dilute the tubulin in Injection Buffer to 2 to 5 mg/ml.

2. Clarify the solution by centrifugation.

3. Inject approximately one-tenth of the cell volume (See Hint #13).

D. Monitoring Polymerization in Pure Labeled Tubulin

1. Use a mixture of labeled and unlabeled tubulin for polymerization. The ratio of labeled to unlabeled will depend on the particular application and on the brightness of the labeled tubulin.

2. Labeled tubulins, especially those labeled to high stoichiometry, exhibit very different properties from unlabeled tubulin. Therefore, use the highest ratio of unlabeled to labeled tubulin that provides signal intensity sufficient for a particular experiment.

Solutions
Injection Buffer (10X)   pH of approximately 7.0
5 mM MgCl2
500 mM Potassium Glutamate (use 2 M Stock)
Low pH cushion   Prepare in 1X BRB80
60% (v/v) Glycerol
Stop Solution   40% (v/v) Glycerol
2X BRB80
100 mM Potassium Glutamate (use 2 M Stock)
Labeling Buffer   40% (v/v) Glycerol
0.1 M Sodium-HEPES, pH 8.6 using NaOH
1 mM EGTA
1 mM MgCl2
High pH Cushion   0.1 M Sodium-HEPES, pH 8.6 using NaOH
1 mM EGTA
1 mM MgCl2
60% (v/v) Glycerol
100 mM GTP
BRB80 (5X)   pH 6.8 with KOH
5 mM MgCl2
400 mM PIPES
Store at 4°C
5 mM EGTA
2 M Potassium Glutamate Stock   pH carefully with KOH to approximately 7.0
2 M Potassium Glutamate
Dye Stock   Prepare in 20 to 100 mM anhydrous DMSO (CAUTION! see Hint #2)
Prepared dye stock such that there is a 10 to 20 molar excess of dye when compared to the tubulin (see Protocol Step #11 and Hint #5)
Dye of choice, such as Tetramethylrhodamine, X-Rhodamine, Fluorescein, Oregon Green, Cy3, Cy5 or C2CF (bis-caged) carboxyfluorescein
Remaining dye solution can be stored at -20°C or -80°C under anhydrous conditions
Purified Tubulin   See Large Scale Tubulin Preparation Protocol
Approximately 50 mg of Tubulin
 
BioReagents and Chemicals
GTP
Potassium Hydoxide
Small Dounce (2 ml)
EGTA
PIPES
Cy5
Cy3
Oregon Green
Sodium Hydroxide
Fluorescein
Magnesium Chloride
X-Rhodamine
Glycerol
Ttetramethylrhodamine
DMSO
Potassium Glutamate
Sodium-HEPES
bis-caged carboxyfluorescein
C2CF
 
Protocol Hints
1. The labeling is performed at high pH to optimize the reaction with the succinimidyl esters.

2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

3. The procedure described below can be scaled down if desired. It is essential to perform all steps involving caged dyes under a safelight in a room well shielded from light. A piece of red acetate sheet taped over a dimly lit lamp is adequate as a safelight. Other dye labelings can be done under room light, minimizing exposure during incubations by using foil.

4. Alternatively, one can add one-half volume of 100% Glycerol instead of DMSO to promote polymerization. This is done as a side-by-side comparison. The contributor of this protocol reports that for reasons that remain unclear using DMSO instead of Glycerol for the first polymerization step appears to increase labeling stoichiometry by approximately 25% for C2CF-SNHS ester. However, for labeling with Rhodamine (Tetramethylrhodamine NHS Ester) and X-Rhodamine, the contributor generally uses Glycerol for polymerization.

5. 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.

6. Estimate the tubulin concentration assuming approximately 70% recovery of the starting tubulin. For dyes such as Cy5 and Cy3, use a 5-pack for labeling approximately 25 mg.

7. For C2CF-SNHS (caged fluorescein), it is best to add the dye in two steps (20 min apart) and label for 60 min at 37°C.

8. Cold Injection Buffer seems to promote more rapid depolymerization than BRB80; therefore, the contributor of the protocol uses Injection Buffer in the depolymerization step for all labeling procedures. For small scale labeling procedures the pellet can be resuspended directly in the centrifuge tube and sonicated gently using a microtip sonicator to speed depolymerization.

9. The pellet should be easy to resuspend.

10. Careful determination of tubulin concentration and labeling stoichiometry can be performed as described in Section B: Quantifying Tubulin Concentration and Labeling Stoichiometry.

11. C2CF-labeled tubulin should be stored at -80°C in a foil-wrapped box.

12. Note that the absorbance of fluorescein is pH-dependent and conjugates with fluorescein should either be diluted into a high pH buffer (approximately 8.8 to 9.0) or the value measured at pH 7.0 multiplied by 1.2.

13. For frog extract studies, use labeled tubulin at one-fortieth to one-two hundredth of the extract tubulin pool (approximately 20 μM).