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'In gel' cleavage with cyanogen bromide for protein internal sequencing
O L Córdoba1, S B Linskens, E Dacci
1Laboratorio Nacional de Investigación, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Journal of Biochemical and Biophysical Methods
|August 1, 1997
Summary
This study introduces an improved method for protein sequencing using in-gel chemical digestion after Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This technique effectively generates internal sequence data from N-terminal blocked proteins for research.
Area of Science:
- Protein Chemistry
- Biochemistry
- Molecular Biology
Background:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a common final step in protein purification for sequencing.
- N-terminal blocked proteins require alternative methods for generating internal sequence data.
Purpose of the Study:
- To propose and validate a novel 'in-gel' digestion procedure for chemical cleavage of proteins purified by SDS-PAGE.
- To enable internal sequence data generation from N-terminal blocked proteins.
Main Methods:
- Proteins purified by SDS-PAGE undergo 'in-gel' chemical cleavage using cyanogen bromide (CNBr).
- Resulting peptides are isolated via a second SDS-PAGE and electroblotted onto PVDF membranes.
- Electroblotted peptides are sequenced using Edman degradation.
Main Results:
- The 'in situ' cleavage profile of proteins was successfully compared with direct chemical digestion in solution.
- The method allows for the obtention of long peptides suitable for sequencing due to limited methionine residues.
- Three standard proteins of varying molecular masses were effectively analyzed.
Conclusions:
- This 'in-gel' digestion and sequencing approach provides a viable strategy for obtaining internal sequence data from proteins purified by SDS-PAGE.
- The method is particularly useful for N-terminal blocked proteins, enhancing protein characterization capabilities.