Sequencing of Proteins from Two-Dimensional Gels by Using in situ Digestion and Transfer of Peptides to Polyvinylidene Difluoride Membranes: Application to Proteins Associated with Sensitization in Aplysia

We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1988-09, Vol.85 (18), p.7008-7012
Main Authors: Kennedy, T. E., Gawinowicz, M. A., Barzilai, A., Kandel, E. R., Sweatt, J. D.
Format: Article
Language:English
Subjects:
Actins
Actins - analysis
Amino Acid Sequence
Amino acids
Analytical, structural and metabolic biochemistry
Animals
Aplysia
Aplysia - analysis
Biological and medical sciences
Central nervous system
Electrophoresis
Electrophoresis, Polyacrylamide Gel
Enzymes
Fundamental and applied biological sciences. Psychology
Gels
General aspects, investigation methods
Homogenization
Membranes, Artificial
Methods
polyvinylidene difluoride
Polyvinyls
Proteins
Proteins - analysis
Rats
Sensitization
Sequencing
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Summary:We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the gel is stained with Coomassie blue and the protein spots of interest are cut out. The in situ protein is digested with Staphylococcus aureus V8 protease in a second polyacrylamide gel and the peptides are separated by one-dimensional polyacrylamide gel electrophoresis. The peptides are then electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Using this method, we have obtained amino acid sequence data for two proteins that are altered after long-term sensitization: actin and Aplysia protein 407. In addition, we have obtained amino acid sequence data for rat protein 425, a protein that appears to be homologous to Aplysia protein 407.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.85.18.7008