Reconstitution and functional analysis of kinetochore subcomplexes.
Methods Cell Biol
; 95: 641-56, 2010.
Article
en En
| MEDLINE
| ID: mdl-20466157
ABSTRACT
Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Cinetocoros
/
Técnicas de Laboratorio Clínico
/
Proteínas Asociadas a Microtúbulos
/
Microtúbulos
/
Huso Acromático
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Cell Biol
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos