Purification of tubulin from limited volumes of cultured cells.
A method was designed to purify tubulin from limited volumes of cultured cells, which can be performed in less than 4 h. The method is based on the preservation of intact microtubule arrays during cell lysis in a large volume of buffer, followed by disassembly of microtubules in a small volume of cold buffer. This allows a good enrichment in tubulin, which is then purified by one cycle of polymerisation/depolymerisation and a cation exchange chromatography. Such a procedure has been employed successfully on suspension-cultured and on adherent HeLa cells. Tubulin obtained was 90% pure, assembly-competent and composed of alpha/beta I and alpha/beta IV isotypes. Microtubules made with this tubulin displayed specific properties such as resistance to dilution, maybe related to their specific dynamic behaviour.
Fourest-Lieuvin
Anne
A
Laboratoire du Cytosquelette, INSERM Unité 366, CEA, Grenoble, France. anne.fourest-lieuvin@cea.fr
D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000737:chemistry; D012680:Sensitivity and Specificity; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification*
D014404:Tubulin