Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE.
Bio Protoc
; 11(16): e4121, 2021 Aug 20.
Article
em En
| MEDLINE
| ID: mdl-34541040
ABSTRACT
Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by sonication, cell supernatants are separated using Tris-Glycine SDS-PAGE, and superfolder GFP (sfGFP) fluorescence is directly visualized within gels. This method is rapid, with sfGFP immediately imaged following SDS-PAGE without staining. Further, sfGFP can be specifically detected in complex samples such as E. coli cell supernatants, proteins run at expected masses, and all steps can be performed at ambient temperature. This strategy is broadly applicable beyond the study of protein splicing and can be used for sensitive and specific visualization of superfolder sfGFP-tagged proteins in-gel.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Bio Protoc
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos