Product Uses Include
Tubulin protein has been purified from porcine brain by an adaptation of the method of Shelanski et al. (1), Further purification to >99% purity was achieved by cation exchange chromatography. Tubulin is supplied as a white lyophilized powder.
Fully active for polymerization, this product is lyophilized with a patented tecnhnology for increased stability and longevity. T240 is stable for 1 year at 4°C desiccated. If your project requires the same batch of tubulin for consistent results, it is highly recommended that the item is purchased in bulk in order to save time and money. This product can be used as a substitute for our highly purified bovine tubulin products (Cat. # TL238, T238 and T237) and behaves in an identical fashion.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >99% pure
Figure 1: A 20 µg sample of T240 protein was separated by electrophoresis on a 10% SDS-PAGE gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02).
One unit of tubulin is defined as 5.0 mg of purified protein (as determined by the Precision Red Advanced Protein Assay Reagent cat. # ADV02). The biological activity of T240 is assessed by a tubulin polymerization assay. The ability of tubulin to polymerize into microtubules can be followed by observing an increase in optical density of the tubulin solution at 340 nm. A 5 mg/ml tubulin solution in General Tubulin Buffer buffer plus 5% glycerol and 1 mM GTP should achieve an OD340 nm reading between 0.75-1.10 in 30 min at 37°C when using a spectrophotometer pathlength of 0.8 cm (180 µl sample volume in a 1/2 area 96-well plate).
It should be noted that tubulin minus glycerol WILL NOT polymerize in G-PEM buffer until very high tubulin concentrations (>10 mg/ml). Even at these concentrations polymerization is comparatively slow. Efficient polymerization at low concentration of tubulin minus glycerol can be achieved by addition of a polymerization stimulating compound, e.g., glycerol, paclitaxel or DMSO.
Shelanski, M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768
Hosono et al., 2012. The murine Gcap14 gene encodes a novel microtubule binding and bundling protein. FEBS Lett. v 586, pp 1426-1430.
Kawakami et al., 2012. LRRK2 Phosphorylates Tubulin-Associated Tau but Not the Free Molecule: LRRK2-Mediated Regulation of the Tau-Tubulin Association and Neurite Outgrowth. PLoS ONE. 7: e30834.
Berezniuk et al., 2012. Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin. J. Biol. Chem. 287, 6503-6517.
Wu et al., 2012. A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice. FASEB J. doi: 10.1096/fj.12-205047.
Ligon et al., 2003. The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization. Mol. Biol. Cell. v 14, pp 1405-1417.
Faivre-Moskalenko and Dogterom, 2002. Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes. Proc. Natl. Acad. Sci. U.S.A. v 99, pp 16788-16793.
Question 1: What is the proper way to store the tubulin to insure maximum stability and activity?
Answer 1: The recommended storage condition for the lyophilized tubulin product is 4°C with desiccant to maintain humidity at <10% humidity. Under these conditions the protein is stable for 6 months. Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months. However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture. Therefore we recommend storing at 4°C. If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. After reconstituting the protein as directed, the concentrated protein in G-PEM buffer should be aliquoted, snap frozen in liquid nitrogen and stored at -70°C (stable for 6 months). NOTE: It is very important to snap freeze the tubulin in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
Question 2: Why does Cytoskeleton recommend the use of general tubulin buffer and GTP for resuspending tubulin?
Answer 2: We recommend resuspending tubulin in general tubulin buffer + GTP to maintain tubulin monomer protein stability and conformation and to provide the necessary components for polymerization. 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 place on ice until right before experimental use.
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