Tip 1: Storage and handling of tubulin

Tubulin is a labile protein. Keep the lyophilized tubulin dry by storing in a desiccator at either 4°C or -70°C. After reconstituting tubulin as directed on the product's datasheet, experiment-sized aliquots must be snap-frozen to liquid nitrogen before storing at -70°C to preserve tubulin's activity. Tubulin should also not be frozen at a concentration below 6 mg/ml. When thawing the aliquots, thaw tubes in a room temperature water bath and immediately place on ice until used. Do not re-freeze tubulin once thawed. Throw away unused thawed tubulin aliquots.

Tip 2: Tubulin polymerization conditions

Temperature
Tubulin polymerization is regulated by temperature. At 37°C tubulin will polymerize into microtubules while at 4°C microtubules will depolymerize to the tubulin subunits. There is a loss of 5% polymer per degree reduction in temperature. It is critical therefore to pay particular attention to temperature throughout the assay. Tubulin should be kept on ice until transferred to the 96 well plate for polymerization at 37°C. The plate reader should be brought to 37°C before beginning assay and the plate and buffers should be pre-warmed to 37°C.

Assay Characterization
In order to achieve reproducible results the researcher must decide on standard conditions of operation. The recommended standard conditions are 2-3 mg/ml tubulin in general tubulin buffer supplemented with 1 mM GTP and 15% glycerol. Using a higher protein concentration will achieve greater polymerization signal which can be useful for detecting inhibitors. Using lower or zero concentrations of glycerol is useful for detecting polymerization enhancing compounds. In the absence of glycerol, tubulin will not polymerize at a concentration below 5 mg/ml except in the presence of an enhancing agent like paclitaxel. Do not use high concentrations of taxol and glycerol together as this combination of enhancers causes aberrant tubulin polymer formations. Conditions can be modified to suit particular requirements. For example, if you wish to search for inhibitors that bind hydrophobic pockets of tubulin you may want to use no glycerol and a higher concentration of tubulin or use microtubule-associated proteins (MAPs; as in MAP-rich tubulin, cat. # ML116) which bind ionically rather than in hydrophobic pockets. Tubulin concentraton and glycerol concentration will also vary based on the purity of tubulin being used. Purer tubulin actually requies more enhancers than tubulin that also contains MAPs since MAPs are natural polymerization enhancers.

FAQ 1: What is the physiological form of tubulin?

Answer 1: The basic physiological unit of tubulin is the heterodimer, composed of 1 alpha and 1 beta subunit (aka alpha and beta tubulin). A true monomer (either 1 alpha or 1 beta subunit) is unstable in vitro and is not known to exist under in vivo conditions. Molecular weight of the heterodimeric subunit is 110,000 daltons, whereas each monomer is 55,000 daltons.

FAQ 2: What kits are available to study my protein of interest?

Answer 2: Cytoskeleton, Inc. offers a variety of kits to study how a protein of interest interacts with and affects tubulin. To examine if a protein is a microtubule binding protein (MAP), we offer the microtubule binding protein spin-down assay biochem kit (Cat. # BK029). This kit provides clear instructions and all the reagents and controls necessary to determine the ability of a given protein to bind to tubulin monomers versus tubulin polymers (microtubules). Tubulin binding can be measured by using a spin down assay where centrifugation is used to separate microtubules from tubulin monomers by differential sedimentation.

We also offer absorbance and fluorescence-based tubulin polymerization assay Biochem Kits (Cat. # BK006P or BK011P, respectively) to follow polymerization kinetics. There are many proteins and drugs that either enhance or inhibit polymerization. These compounds can be characterized by their effect on one or more of the three stages of tubulin polymerization: nucleation, growth, and steady-state equilibrium. A compound’s effects on depolymerization can also be evaluated.

Additionally, the microtubule/tubulin in vivo assay kit (Cat. # BK038) offers a straightforward method for quantitating the in vivo ratio of tubulin polymers to monomers. This kit allows the researcher to easily analyze changes in tubulin monomers and polymers induced by their protein of interest in cell or tissue samples.

To examine if your protein of interest affects how microtubules interact with kinesins, Cytoskeleton also offers ATPase assays that quantify the levels of inorganic phosphate production by microtubule-activated kinesin ATPase activity. An end-point (Cat. # BK053) and kinetic (Cat. # BK060) kit is offered.

To examine site-specific competition for binding sites on tubulin, there are several technical tips available from tservice@cytoskeleton.com . 

FAQ 3: What assays are available to screen compounds for anti-microtubule activity?

Answer 3: Cytoskeleton, Inc. supplies multiple kits that can be used to examine a compound’s anti-microtubule activity. We offer absorbance and fluorescence-based tubulin polymerization assay Biochem Kits (Cat. # BK006P or BK011P, respectively) to follow polymerization kinetics. The polymerization kits can be used to evaluate a compounds’s ability to induce microtubule depolymerization or impair polymerization. These microtubules can then be incubated with compounds and changes in the length and number of microtubules can be quantified. Additionally, the microtubule/tubulin in vivo assay kit (Cat. # BK038) offers a straightforward method for quantitating the in vivo ratio of tubulin polymers to monomers. This kit allows the researcher to easily analyze changes in tubulin monomers and polymers induced by various treatment conditions in cell or tissue samples.