Electroelution of Proteins from Polyacrylamide Gels
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One- or two-dimensional polyacrylamide gel electrophoresis (PAGE) is one of the most versatile methods for protein separation. The resolving power of a two-dimensional O’Farrell gel (1 and see Chapter 20–22) cannot be reached with today’s high-performance liquid chromatographic techniques unless specific affinity interactions are being exploited for protein purification. In combination with electroblotting, analytical sodium dodecyl sulfate (SDS)-PAGE is often used to prepare proteins for N-terminal sequencing (2). Recent developments have made it possible to fragment proteins electroblotted onto membranes enzymatically and to obtain sequence information from proteins that are not amenable to N-terminal Edman degradation (3). For the purpose of obtaining protein sequence information, electroblotting has almost completely replaced electroelution of proteins from the polyacrylamide matrix. The reason for this is that electroeluted proteins are contaminated with Coomassie blue, buffer salts, and large amounts of SDS, making it necessary to desalt samples before subsequent Edman degradation. So why electroelution?
KeywordsSodium Dodecyl Sulfate Coomassie Blue NANOpure Water Hydrophilic Interaction Chromatography Ammonium Hydrogen Carbonate
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