Summary
A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed 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 sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
R. C. Allen, C. A. Saravis, H. G. Maurer, Gel Electrophoresis and Isoelectric Focusing of Proteins, De Gruyter, Berlin 1984.
R. Aebersold, D. Teplow, L. E. Hood, S. B. H. Kent, J. Biol. Chem.261, 4229–4238 (1986).
J. Vandekerchhove, G. Bauw, M. Puype, J. Van Damme, M. van Montagu, Eur. J. Biochem.152, 9–19 (1985).
P. Matsudaira, J. Biol. Chem.262, 10035–10038 (1987).
C. Eckerskorn, W. Mewes, H. Goretzki, F. Lottspeich, Eur. J. Biochem.176, 509–519 (1988).
NBFR-PIR Protein Sequence Database, Release 16, 1988, Document PRTAPE-0388.
U. K. Laemmli, Nature227, 680–685 (1970).
P. H. O'Farrell, J. Biol. Chem.250, 4007–4021 (1975).
F. Lottspeich, J. Chromatogr.326, 321–327 (1985).
H. Homann, Thesis, Universität München (1988).
W. J. Neubert, P. H. Hofschneider, Virology125, 445–449 (1983).
C. Eckerskorn, P. Jungblut, J. Klose, F. Lottspeich, Electrophoresis9, 830–838 (1988).
R. Aebersold, J. Leavitt, L. E. Hood, S. B. H. Kent, Proc Natl. Acad. Sci. USA84, 6970–6974 (1987).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Eckerskorn, C., Lottspeich, F. Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix. Chromatographia 28, 92–94 (1989). https://doi.org/10.1007/BF02290390
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02290390