Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin Polymerization Protocol using GTP and GMPCPP

A. GMPCPP Seed Formation (see Tip #1)

1. To form short GMPCPP seeds, mix unlabeled Tubulin and labeled Tubulin (1 to 3 mg/ml final) at an appropriate ratio in 1X BRB80 with 1 mM DTT and 0.5 to 1 mM GMPCPP on ice.
2. Incubate at 0°C for 5 to 10 min.
3. Clarify by centrifuging the mixture for 5 min in TLA100 rotor at 90,000 rpm (360,000 X g) at 2°C.
4. Freeze the supernatant (CPP Tubulin) in 5 to 10 μl aliquots in Liquid Nitrogen and store at -80°C.
5. Transfer a tube of CPP Tubulin from the freezer to a 37°C bath.
6. Incubate for 15 to 20 min at 37°C.
7. Dilute to 150 to 200 μl with warm BRB80 with 1 mM DTT.
8. Centrifuge the seeds in a TLA100 rotor at 90,000 rpm (360,000 X g) for 5 min at 25 to 30°C.
9. Discard the supernatant and resuspend the pellet in 1 to 2 volumes of BRB80 with 1 mM DTT. This process removes free CPP and any unpolymerized Tubulin.

B. Clarification of Prepared Tubulin

1. Mix unlabeled Tubulin and labeled Tubulin at an appropriate ratio (1 to 3 mg/ml final) in 1X BRB80 with 1 mM DTT and 1 mM GTP on ice (see Tips #2 and #3).
2. Incubate for 5 min at 0°C.
3. Centrifuge the mixture for 5 min using a TLA100 rotor at 90,000 rpm (360,000 X g) at 2°C.

C. Polymerization of Tubulin

1. Collect the supernatant from Step #B3 and incubate at 37°C for 1 min.
2. Add a small volume of the GMPCPP seeds made in Section A (approximately 0.05 to 0.02 volume) after the polymerization mix has been at 37°C for 1 min (see Tip #4). Axonemes or centosomes can be used as nucleating structures.
3. Continue incubation at 37°C. If the Tubulin concentration is 2 mg/ml or higher, assembly will proceed rapidly to steady state (approximately 30 min). If the concentration is less than 2 mg/ml, nucleation can be limiting and the precise kinetics of approach to steady state is difficult to predict. In this case, it will depend on the amount of active Tubulin in the mix. (see Tip #5)

Tubulin Polymerization Buffers

1 mM DTT BRB80 Solution (1X)    (Generally kept as a 5X stock and stored at 4°C) 80 mM PIPES 1 mM EGTA 1 mM MgCl2 pH 6.8 100 mM GMPCPP 100 mM GTP

Materials

Tips for Protocol