Table I.
Microplate Reaction Volumes
| Component | Stock | Volume added (μl) | Total volume (μl) | Final |
|---|---|---|---|---|
| Brain tubulin | 100 μM | 1 | 2 μM | |
| Pipes buffer | 0.1 M | 47 | 0.1 M | |
| Drug (in PM Buffer) | Varied | Up to 2 | 4% v/va | |
| DMSO | 100% | 2 minus drug volumea | 4% v/va | |
| Initial volume | – | – | 50b | – |
| DTNB | 3 mM | 5 | 0.3 mM | |
| Total reaction volume | – | – | 55c | – |
| GuHCl | 5 M | 25 | 80d | 1.6 M |
DMSO, dimethyl sulfoxide; DTNB, 5′,5′-dithiobis(2-nitrobenzoate); GuHCl, guanidine hydrochloride.
a
Total DMSO + ligand should be 4% of total volume. Add additional DMSO to bring total DMSO + ligand up to 2 μl if drug addition is below 2 μl.
b
Tubulin and drug/DMSO mixture should be allowed to equilibrate for 10 min at room temperature.
c
After DTNB addition, mix carefully and begin gathering A412 for 40 min.
d
GuHCl can be added after completing A412 step to ensure equal protein concentration (through reacted thiol content) across each reaction.