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.
Incubate at 0°C for 5 to 10 min.
Clarify by centrifuging the mixture for 5 min in TLA100 rotor at 90,000 rpm (360,000 X g) at 2°C.
Freeze the supernatant (CPP Tubulin) in 5 to 10 μl aliquots in Liquid Nitrogen and store at -80°C.
Transfer a tube of CPP Tubulin from the freezer to a 37°C bath.
Incubate for 15 to 20 min at 37°C.
Dilute to 150 to 200 μl with warm BRB80 with 1 mM DTT.
Centrifuge the seeds in a TLA100 rotor at 90,000 rpm (360,000 X g) for 5 min at 25 to 30°C.
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
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).
Incubate for 5 min at 0°C.
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
Collect the supernatant from Step #B3 and incubate at 37°C for 1 min.
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.
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
1. In order to surmount the nucleation barrier in the polymerization assay and thereby specifically assay Tubulin elongation, GMPCPP seeds can be added to the incubation in Step C2 GMPCPP is the best current GTP analog for Tubulin polymerization. Its major limitation is lack of commercial availability.
2. For example, for a dim polymerization mix use 1 part labeled Tubulin to 10 parts unlabeled Tubulin. For bright polymerization mix, use 1 part labeled Tubulin to 2 parts unlabeled Tubulin.
3. The first three steps of incubating on ice and centrifuging to clarify the solution are especially important when polymerization includes GMPCPP and/or highly labeled fluorescent Tubulins. Also, if thawed labeled Tubulins are used for microinjection, this centrifugation step is recommended to clarify the solution.
4. Given the approximately 10-fold higher affinity of Tubulin for GTP versus GMPCPP, the amount of GMPCPP added from a seed mix at these dilutions is insignificant. Therefore, you can add seeds directly into a Polymerization Mix without dilution/sedimentation/resuspension.
5. If the purpose is labeling or recycling the Tubulin, polymerization is promoted by addition of DMSO (CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions) or Glycerol. After 2 min incubation at 37°C, add 0.5 volume of Glycerol (It may be more convenient to weigh out the Glycerol, using the density for Glycerol which is 1.26 g/ml). Mix the Glycerol into the polymerization reaction by gentle vortexing. Or add DMSO to a final concentration of 10% to the polymerization incubation after 2 min at 37°C.