Vertical Agarose Gel Electrophoresis for Analysis of High Molecular Weight Proteins

SDS-PAGE does not effectively separate proteins larger than 200 kDa. High molecular weight proteins have difficulties moving and entering small pores of polyacrylamide gel. Vertical agarose gel electrophoresis can be a good alternative to separate and resolve large molecular weight proteins. Resolved proteins can be visualized on agarose using different stains. In addition, vertical agarose gel electrophoresis allows efficient transfer of resolved protein to a membrane for further analysis.

The following peer-reviewed articles provide standardized protocols for 1. Preparing vertical agarose gel, 2. Loading and running protein samples and 3. Transfer of resolved proteins from gel to a membrane for further analysis.

  • Greaser & Warren, 2019. Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose. Methods Mol Biol. 1855, 203-210. PMID-30426419; Full Text Link: springer 
  • Greaser & Warren, 2015. Method for resolution and western blotting of very large proteins using agarose electrophoresis. Methods Mol Biol. 1312, 285-91. PMID-26044011; Full Text Link: springer 
  • Greaser & Warren, 2012. Protein electrophoresis in agarose gels for separating high molecular weight proteins. Methods Mol Biol. 869, 111-8. PMID-22585481; Full Text Link: springer
  • Greaser & Warren, 2009. Efficient electroblotting of very large proteins using a vertical agarose electrophoresis system. Methods Mol Biol. 536, 221-7. PMID-19378061; Full Text Link: springer
  • Warren et al., 2003. Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins.   Electrophoresis. 24(11), 1695-702. PMID-12783444; Full Text Link: wiley

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