Rapid purification of tubulin from tissue and tissue culture cells using solid-phase ion exchange.
Tubulin can be purified from tissue or tissue culture cell starting material in less than 6 h using a combination of a solid-phase ion exchanger and one cycle of temperature-dependent polymerization and depolymerization. The use of a solid-phase ion exchanger (MemSep DEAE) greatly reduces the time required for the ion exchange step compared to conventional exchangers. The use of sodium glutamate to elute tubulin from the exchanger stabilizes the protein and facilitates the final polymerization step of the purification. The procedure can be performed successfully with less than 100 mg of tissue culture cell protein as starting material. The final material is > 90% pure and active as judged by sodium dodecyl sulfate-gels and by binding of the bicyclic colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-tropone.
Sackett
D L
DL
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D002457:Cell Extracts; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005260:Female; D009363:Neoplasm Proteins / Q000302:isolation & purification; D011108:Polymers; D051381:Rats; D017207:Rats, Sprague-Dawley; D012970:Sodium Glutamate; D012995:Solubility; D013696:Temperature; D013997:Time Factors; D014404:Tubulin / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D009363:Neoplasm Proteins; D011108:Polymers; D014404:Tubulin; D012970:Sodium Glutamate