Cat. # BK010

Product Uses Include

• Screen for dicotyledon targeted herbicides.
• Basic research to measure effects of proteins and compounds on polymerization activity.
• Basic research plant tubulin polymerization studies.
• Source of plant tubulin for monomer or polymer (microtubule) binding studies (similar to BK029).
  
  

Introduction
This assay is a one step procedure for performing plant tubulin polymerization assays. Polymerization is followed by fluorescence enhancement due to the incorporation of a fluorescent reporter into microtubules as polymerization occurs. It is an adaptation of an assay originally described by Bonne, D. et al. (1) for bovine brain tubulin. By calculating Vmax of polymerization in AFU/min you can determine the specific activity of a compound or protein on plant tubulin polymerization.

Example results
Compounds or proteins that interact with tubulin will often alter one or more of the characteristic phases of polymerization.  For example, Figure 1 shows the effect of adding the anti-mitotic drug paclitaxel to the standard polymerization reaction.  A 3 µM concentration of paclitaxel eliminates the nucleation phase and enhances the Vmax of the growth phase.  Thus, one application of this assay is the identification of novel anti-mitotic compounds that enhance polymerization and stabilize microtubules.  Figure 1 also shows the effect of adding the tubulin binding drug, trifluran.  At 3 µM final concentration, trifluran causes a drastic decrease in Vmax and reduction in final polymer mass. 

Figure 1. Plant Tubulin polymerization using the fluorescence based tubulin polymerization assay (Cat.# BK010).

Tubulin was incubated alone (Control), with Paclitaxel or Trifluran. Each condition was tested in duplicate. Polymerization was measured by excitation at 360 nm and emission at 420 nm.  The three Phases of tubulin polymerization are marked for the control polymerization curve; I: nucleation, II: growth, III: steady state equillibrium.

To obtain dose response curves we utilized the Vmax values and plotted these against the log10 concentration of compound (see Figure 2). For quantitative comparison purposes we determined the IC50 or EC50 value by the 50% vertical intercept of the dose response curves. As shown in Table 1 there is a wide range of IC50 concentrations when comparing the two tubulin sources (i.e. plant versus neuronal tubulins).

Figure 2. Dose response curves

Table 1. IC50 values

In  Table 1, the results are compared with bovine brain tubulin using the same microassay. Pendimethalin is >28 times more effective at inhibiting soybean tubulin polymerization compared to neuronal tubulin. Whereas Trifluralin is >52 times more effective against soybean tubulin. These values are represented by the ratio called the Tubulin Ligand Index ratio (TLI ratio; IC50 bovine brain tubulin :  IC50 plant tubulin) which compares activities against neuronal and soybean tubulin. These preliminary results indicate that this new microassay may be used to compare known compounds for their efficacy on plant and animal tubulins. For more information about developing other tubulin sources contact customer service at cserve@cytoskeleton.com or call USA-303-322-2254. Custom screening for with various tubulins to create TLI values is also available.

Kit contents
The kit contains sufficient material for 24 assays in 10 µl format. The following components are included:

1. >90% pure tubulin (Cat. # TP005 [not sold separately])
2. General tubulin buffer with fluorescent reporter
3. Compound dilution buffer
4. GTP solution (Cat. # BST06)
5. Paclitaxel positive control (Cat. # TXD01)
6. Small volume 384-well plate.
7. Manual with detailed protocols and extensive trouble shooting guide