Tubulin (porcine brain, >99% pure)

On Arrival: 4°C

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&rsquo, 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.

IC50 & EC50 determinations for anti-tubulin ligands.
Microtubule binding studies
Tubulin monomer binding studies
HDAC6 studies
Microtubule activated kinesin ATPase assays

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 OD_{340 nm} reading between 0.75-1.10 in 30 min at 37CWhen using a spectrophotometer pathlength of 0.8 cm (180 ul 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.