Materials

1.Drop frozen 2x cycled tubulin in a polymerization-promoting buffer.Stored at -80℃

2.GTP,100 mM stock,titrate to pH ~7.Need 2 ml.

3.Waterbath,set at 37℃

4.SS34 rotor,5 ml and 15 ml tubes and adaptors.Bring the rotor,adaptors,and tubes to ~37℃ by soaking in warm water.If possible,keep a separate centrifuge and SS34 rotor at 4℃.

5.PEM buffer: 0.1 M PIPES,1.0 mM EGTA,0.5 mM MgCl2,pH 6.9,prepare 50 ml.Keep ~20 ml at 37℃ and the rest on ice.

6.DMSO.

7.TRSE,Molecular Probes C-1171.Prepare fresh 10 mg/ml stock in DMSO.Need 2.5 mg per 50 mg of tubulin.

8.Potassium glutamate,1 M.Prepare 1 M glutamic acid and titrate with KOH to pH 6.9.

9.Injection Buffer: 50 mM potassium glutamate,0.5 mM MgCl2,pH 6.5-6.7.Prepare 10 ml.Keep ~5 ml at 37℃ and the rest on ice.

10.1 M PIPES,pH 6.9.Need 5-10 ml.

Procedure

1.Weigh out drop frozen pellets of 2x cycled tubulin to obtain ~50 mg of tubulin.Thaw in a water bath.

2.Add an equal volume of 1 M PIPES (volume in mls equals tubulin weight in grams).Mix well.Add GTP to 1 mM and DMSO to 10% and mix well.Incubate at 37℃ for 10 min.The solution should turn turbid and viscous.

3.Centrifuge in a SS34 rotor at 18,000 rpm,37℃ for 20 min.Keep the rotor warm in an incubator afterwards.

4.Discard the supernatant.Resuspend pellets in 3 ml of ice cold PEM buffer by gentle pipeting.Measure the total volume.

5.Incubate at 0℃ for 10 min.

6.Add GTP to 1 mM,and DMSO very slowly while stirring to 10%.Incubate at 37℃ for 10 min.Prepare the TRSE stock solution.

7.Transfer the tubulin solution to a vial with a stir bar.While stirring vigorously,add very slowly 250 microliters of the TRSE stock.

8.Incubate at 37℃ for 5 min,then add potassium glutamate to 5 mM to stop the reaction.

9.Centrifuge in a SS34 rotor at 18,000 rpm,37℃ for 20 min.Chill down the rotor afterwards.

10.Rinse pellet quickly with 2x2 ml warm PEM buffer.Resuspend pellet in 2 ml of cold PEM buffer (solution appears opaque).

11.Incubate at 0℃ for 10 min.

12 Centrifuge in a SS34 rotor at 18,000 rpm,4℃ for 20 min.Warm up the rotor afterwards.

13.Collect supernatant and measure its volume.Bring GTP to 1 mM and DMSO to 10%.

14.Incubate at 37℃ for 10 min.

15.Centrifuge in a SS34 rotor at 18,000 rpm,37℃ for 20 min.Chill down the rotor afterwards.

16.Rinse the pellet quickly with 2x2 ml warm PEM buffer.Resuspend pellet in 1 ml of cold PEM buffer by gentle pipeting.

17.Cycle tubulin as in steps 11 through 15.Use 5 ml centrifuge tubes for smaller volumes.

18.Rinse pellet twice,quickly,with warm injection buffer.Resuspend pellet in ~500-800 microliters of ice cold injection uffer.Incubate at 0℃ for 10 min.

19.Centrifuge in a SS34 rotor at 18,000 rpm,4℃ for 20 min

20.Drop freeze the labeled tubulin.

21.Final tubulin concentration is determined by the Lowry assay.Take a 10-20μl aliquot and dilute with 400μl buffer.Read OD at 556 nm and calculate dye concentration based on the molar extinction coefficient of 50,000.D/P should be around 0.75 per tubulin monomer.