Zero-length crosslinking procedure with the use of active esters.
Anal Biochem
; 185(1): 131-5, 1990 Feb 15.
Article
en En
| MEDLINE
| ID: mdl-2344038
ABSTRACT
A two-step zero-length crosslinking procedure for studying protein-protein complexes has been developed. One component of a complex is briefly incubated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide resulting in the conversion of some of the protein carboxyls into succinimidyl esters. The reaction is stopped by addition of beta-mercaptoethanol and other interacting proteins are then added. Crosslinking arises from substitution of lysine epsilon-amino groups of these proteins for the succinimidyl moieties during a 1- to 2-h incubation period. The advantage of this method versus one-step zero-length crosslinking is that only one component of the complex is exposed to the crosslinker, which eliminates complications arising from the formation of crosslinks among several proteins of a multicomponent complex. Furthermore, crosslinks can be formed even in the presence of reagents, such as dithiothreitol and EDTA, that would interfere with direct crosslinking with EDC.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas
/
Reactivos de Enlaces Cruzados
Límite:
Animals
Idioma:
En
Revista:
Anal Biochem
Año:
1990
Tipo del documento:
Article