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Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose

Aebersold, Ruedi H. and Leavitt, John and Saavedra, Raul A. and Hood, Leroy E. and Kent, Stephen B. H. (1987) Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proceedings of the National Academy of Sciences of the United States of America, 84 (20). pp. 6970-6974. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:AEBpnas87

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Abstract

We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.


Item Type:Article
Additional Information:© 1987 by the National Academy of Sciences. Contributed by Leroy E. Hood, May 20, 1987. We thank Wade Hines and Gary Pipes for expert assistance. This research was supported by National Science Foundation Grant DMB 8500298, by research grants from the Monsanto Company and Upjohn Pharmaceuticals Co., and by National Cancer Institute Grants CA 34763 (J.L.) and CA 32911. R.H.A. was the recipient of a fellowship from the Swiss National Science Foundation. R.A.S. is a postdoctoral fellow of the National Multiple Sclerosis Society.
Subject Keywords:protein sequence analysis; electroblotting; peptide mapping; Edman degradation
Record Number:CaltechAUTHORS:AEBpnas87
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:AEBpnas87
Alternative URL:http://www.pnas.org/cgi/content/abstract/84/20/6970
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7570
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 Mar 2007
Last Modified:26 Dec 2012 09:33

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