反应详情

Enzymatic activity of the Eg5 motor and effects of inhibitors was measured using a malachite green assay, which measures phosphate liberated from ATP, and has been used previously to measure the activity of kinesin motors (Hackney and Jiang, 2001). Enzyme was recombinant HsEg5 motor domain (amino acids 1-369-8His) and was added at a final concentration of 6 nM to 100 μl reactions. Buffer consisted of 25 mM PIPES/KOH, pH 6.8, 2 mM MgCl2, 1 mM EGTA, 1 mM dtt, 0.01% Triton X-100 and 5 μM paclitaxel. Malachite green/ammonium molybdate reagent was prepared as follows: for 800 ml final volume, 0.27 g of Malachite Green (J. T. Baker) was dissolved in 600 ml of H2O in a polypropylene bottle. 8.4 g ammonium molybdate (Sigma) was dissolved in 200 ml 4N HCl. The solutions were mixed for 20 min and filtered through 0.02 μm filter directly into a polypropylene container. 5 μl of compound diluted in 12% DMSO was added to the wells of 96 well plates. 80 μl of enzyme diluted in buffer solution above was added per well and incubated with compound for 20 min. After this pre-incubation, substrate solution containing 2 mM ATP (final concentration: 300 μM) and 6,053 μM polymerized tubulin (final concentration: 908 nM) in 15 μl of buffer were then added to each well to start reaction. Reaction was mixed and incubated for an additional 20 min at room temperature. The reactions were then quenched by the addition of 150 μl malachite green/ammonium molybdate reagent, and absorbance read at 650 nanometers exactly 5 min after quench using a Spectramax Plus plate reader (Molecular Devices). Data was graphed and IC50s calculated using ExCel Fit (Microsoft).

1. additionwas added at a final concentration of 6 nM to 100 μl reactions
2. additionThe solutions were mixed for 20 min
3. filtrationfiltered through 0.02 μm
4. filtrationfilter directly into a polypropylene container
5. addition5 μl of compound diluted in 12% DMSO
6. additionwas added to the wells of 96 well plates
7. addition80 μl of enzyme diluted in buffer solution
8. additionabove was added per well and
9. additionAfter this pre-incubation, substrate solution containing 2 mM ATP (final concentration: 300 μM) and 6,053 μM
10. custompolymerized tubulin (final concentration: 908 nM) in 15 μl
11. additionof buffer were then added to each well
12. customreaction
13. customReaction
14. additionwas mixed
15. waitincubated for an additional 20 min at room temperature
16. customThe reactions were then quenched by the addition of 150 μl malachite green/ammonium molybdate reagent, and absorbance
17. customread at 650 nanometers exactly 5 min
18. customafter quench