Tubulin Glycerol Buffer

Question 1: Can the tubulin glycerol buffer be used to separate microtubules from non-polymerized tubulin and/or non-MAP proteins?

Answer 1: Yes, the tubulin glycerol buffer ((Cat. #BST05, aka cushion buffer) can be used undiluted as a 60% glycerol cushion. Microtubules should be layered onto a 5X volume of cushion buffer and centrifuged at 100, 000 x g for 30 minutes. The microtubules will pellet through the cushion and leave any unpolymerized tubulin and non-MAP proteins at the cushion buffer interface. The cushion can be gently removed and the microtubule pellet resuspended and analyzed as required. It is important to note that the cushion buffer should be supplemented with reagents that are necessary for tubulin/microtubule stability (e.g., 1 mM GTP and 10 &mu, M taxol).

Question 2: What is the optimal concentration of glycerol to include for enhancement of polymerization?

Answer 2: Glycerol is often added to a final concentration of 5 - 10% to enhance polymerization, however, glycerol is not necessary for the maintenance of biologically active tubulin. We also do not recommend adding high concentrations of glycerol (10-20%) with taxol. Either glycerol or taxol alone should be used to enhance tubulin polymerization. As tubulin concentrations are reduced (<, 2 mg/ml), the glycerol concentration can be increased up to 20%, but we do not recommend using above this concentration. A high glycerol concentration can lead to a ceiling effect and mask other enhancers, so if testing polymerization enhancers, we recommend omitting glycerol in the experimental drug conditions. Glycerol is ideal as a treatment standard against which test enhancers can be evaluated.