Drugs and Buffers

Tubulin polymerization is highly dependent on buffer conditions, it is therefore vital to use the right buffers. For example, tubulin polymerization is highly dependent on the concentration of magnesium chloride which should be between 1 and 5 mM (see our "About tubulin" pages for more information). We provide pre-mixed buffers for high reproducibility between experiments.

Cytoskeleton also provides the microtubule stabilizing compound paclitaxel, which is provided in packs of ten vials for consistent and economical use. For more information click on the datasheet below. 

Cytoskeleton's products have been cited hundreds of times over the past 18 years. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.

Paclitaxel: 2mM (Cat. # TXD01)

Kosturko, L. D., Maggipinto, M. J., D'Sa, C., Carson, J. H. and Barbarese, E. (2005). The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2. Mol. Biol. Cell 16, 1938-1947.

Teckchandani, A. M., Birukova, A. A., Tar, K., Verin, A. D. and Tsygankov, A. Y. (2005). The multidomain protooncogenic protein c-Cbl binds to tubulin and stabilizes microtubules. Exp. Cell Res. 306, 114-127.

Nair, K. S., Hanson, S. M., Kennedy, M. J., Hurley, J. B., Gurevich, V. V. and Slepak, V. Z. (2004). Direct binding of visual arrestin to microtubules determines the differential subcellular localization of its splice variants in rod photoreceptors. J. Biol. Chem. 279, 41240-41248.

Wagner, O. I., Ascano, J., Tokito, M., Leterrier, J. F., Janmey, P. A. and Holzbaur, E. L. (2004). The interaction of neurofilaments with the microtubule motor cytoplasmic dynein. Mol. Biol. Cell 15, 5092-5100

Ligon, L. A., Shelly, S. S., Tokito, M. and Holzbaur, E. L. (2003). The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization. Mol. Biol. Cell 14, 1405-1417.

Benink, H. A., Mandato, C. A. and Bement, W. M. (2000). Analysis of cortical flow models in vivo. Mol. Biol. Cell 11, 2553-2563.

Korinek, W. S., Copeland, M. J., Chaudhuri, A. and Chant, J. (2000). Molecular linkage underlying microtubule orientation toward cortical sites in yeast. Science 287, 2257-2259.

Question 1:  Can I use PBS to resuspend tubulin protein?

Answer 1: Yes, tubulin can be resuspended in PBS, but the tubulin protein will be unstable and will not polymerize.  For resuspension, we recommend using a general tubulin buffer (Cat. # BST01-001) which consists of 80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 7.0, supplemented with 1 mM GTP (Cat. # BST06-001).  Tubulin requires GTP and magnesium ions for proper stability and conformation, even in its monomeric state.  GTP is also required for the polymerization process as its hydrolysis during tubulin polymerization is necessary for polymerization to occur.  EGTA is a chelator of calcium which is a potent inhibitor of tubulin polymerization.  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 and does not need to be included when reconstituting and storing tubulin.  When aliquoting reconstituted tubulin for storage, it is essential to aliquot and snap-freeze tubulin in liquid nitrogen at a concentration of >6 mg/ml to preserve tubulin’s biological activity.  Then the aliquots should be stored at -70°C.  When thawing the aliquots, thaw rapidly in a room temperature water bath and immediately place on ice, keep on ice until right before use.

 

Question 2:  What is a good concentration of taxol to use to stabilize microtubules?

Answer 2: Taxol, also known as Paclitaxel (Cat. # TXD01), is used to stabilize microtubules.  Paclitaxel binds to an internal region on microtubules and stabilizes them against depolymerization both in vivoand in vitro. The compound should be added to microtubules to a final concentration of 10-20 uM (i.e., a 1:200 - 1:100 dilution of the 2 mM stock solution as directed on the datasheet (Cat. # TXD01) i.e. containing an intermediate sub-dilution to avoid precipitation problems.  Paclitaxel-stabilized microtubules will be stable at room temperature for 3 days.  When resuspending the taxol, use either DMSO or ethanol.  Methanol should be avoided as the taxol will undergo trans-esterification in this solvent.