A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization.
Electrophoresis
; 31(3): 440-7, 2010 Jan.
Article
em En
| MEDLINE
| ID: mdl-20119951
ABSTRACT
To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A "Counter Free-Flow" elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of approximately 10-150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 microL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 microg per channel and reduced resolution.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Eletroforese em Gel de Poliacrilamida
/
Ensaios de Triagem em Larga Escala
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos