Analysis of purified tubulin in high concentration of glutamate for application in high throughput screening for microtubule-stabilizing agents.
High-throughput screening (HTS) is a powerful approach for the discovery of potential and effective drugs from a number of candidates. The dynamics of tubulin assembly and disassembly in vitro are an important target for the screening of anti-tumor agents. However, previously described methods are not amenable for HTS. In this paper, we compared preparation methods of tubulin and suggest a combination method, i.e., one cycle of assembly and disassembly following ion-exchange chromatography using high concentrations of glutamate to increase the recovery of tubulin with high purity. The resultant highly purified microtubule-associated proteins-free tubulin in high glutamate solution was directly employed in tubulin polymerization assays. Our results indicate that the system is feasible and practicable as a model for HTS for microtubule-stabilizing agents.
Dong
Hui
H
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
Li
Yue-Zhong
YZ
Hu
Wei
W
D002848:Chromatography, DEAE-Cellulose; D020650:Combinatorial Chemistry Techniques; D004353:Drug Evaluation, Preclinical; D018698:Glutamic Acid / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects*; D011108:Polymers / Q000138:chemical synthesis; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification
D011108:Polymers; D014404:Tubulin; D018698:Glutamic Acid; D006160:Guanosine Triphosphate