Aebersold, Ruedi H. and Teplow, David B . and Hood, Leroy E . and Kent, Stephen B . H. (1986) Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis. Journal of Biological Chemistry, 261 (9). pp. 4229-4238. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:AEBjbc86
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We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein- containing bands or spots are cut out and inserted directly into a gas- phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150- pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.
|Additional Information:||Copyright © 1986 by the American Society for Biochemistry and Molecular Biology. Received for publication, July 22, 1985. The contributions of Dr. Lloyd Smith, for suggesting the use of fluorescent dyes, and the expert technical contributions of John Kim, Eva Lujan, Wade Hines, and Chin Sook Kim are gratefully acknowledged.|
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|Deposited By:||Lindsay Cleary|
|Deposited On:||14 Sep 2006|
|Last Modified:||26 Dec 2012 09:02|
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