Caki-1 tumor tubulin

AT

1-2 Weeks

IC50 & EC50 determinations for anti-tubulin ligands.
Characterization of tubulin binding proteins.

The biological activity of TM001 is assessed by a tubulin polymerization assay. The ability of tubulin to polymerize into microtubules can be followed by observing an increase in fluorescence using 10?M Dapi in the G-PEM buffer plus 10% glycerol with 2mg/ml tubulin (see Figure 3). Under these experimental conditions the fluorescence will increase by 15-20% over 15 minutes at 37C (see Figure 3). The assay volume is 10 ?l and a low volume 384well plate is used (Corning # 3676).

1. Place one TM001 vial on ice.
2. Resuspend in 125 ?l of G-PEM plus 10% glycerol and 0.05%Triton X-100.
3. Prepare compounds for screening at 5x concentration in PEMplus 5% DMSO.
4. Pipette 2 ?l compound into each well of a pre-warmed 37C plate.
5. Pipette 10 ?l of tubulin solution into each well and start theplate reader protocol. Measure tubulin polymerization by takingreadings every 30 seconds for 30 min.
6. Figure 3 shows the results of polymerizing TM001 under the conditions described above.
Important Technical Notes: When Working with Tubulin protein
1. Any buffer containing GTP should be kept on ice and used within 1-2h after addition of GTP as GTP will hydrolyze overtime. Unused GTP supplemented buffer should be discarded.
2. Tubulin is a labile protein and should be used immediately after thawed or snap frozen into appropriate aliquots (see Storage section). Freeze/thaw cycles should be avoided. Keep tubulinon ice prior to beginning the polymerization reaction.
3. Temperature is an extremely important parameter for tubulin polymerization. Temperatures cooler than 37C will significantly decrease the rate and final OD340nm reading of a polymerization reaction. If tubulin is aliquoted into a cool plate (or room temperature plate) there will be a much longer nucleation phase (Figure 3).
4. Polymerization conditions can be altered to optimize a given assay requirement. For example, to examine polymerization enhancers such as taxol, it is recommended to reduce the tubulin concentration to 1 3 mg/ml and polymerize in buffer without glycerol. These conditions will result in a very slow andshallow polymerization curve for the "no compound" control. In this case, efficient polymerization is achieved by addition of an enhancer such as taxol (5 - 10 ?M final concentration).