Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.
agarose native gel
two-dimensional gel electrophoresis
western blotting
Journal
Electrophoresis
ISSN: 1522-2683
Titre abrégé: Electrophoresis
Pays: Germany
ID NLM: 8204476
Informations de publication
Date de publication:
09 2023
09 2023
Historique:
revised:
22
05
2023
received:
21
10
2022
accepted:
23
05
2023
medline:
14
9
2023
pubmed:
9
6
2023
entrez:
9
6
2023
Statut:
ppublish
Résumé
A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.
Identifiants
pubmed: 37294166
doi: 10.1002/elps.202200255
doi:
Substances chimiques
Sepharose
9012-36-6
Proteins
0
Gels
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1446-1460Informations de copyright
© 2023 Wiley-VCH GmbH.
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