Automated Chemical Sequencers
Chemical protein sequence analysis is performed on an automated Applied Biosystems (ABI 470) protein sequencer. The instrument performs a chemical reaction known as the Edman degradation in which the amino acids are derivatized and removed sequentially from the N-terminus of the peptide or protein. The amino acid derivatives are then identified after HPLC separation allowing the amino acid sequence of the protein/peptide to be deduced. For the determination of the N-terminal sequence of a purified protein/peptide 5-100 pmol of material is required. Residues can be identified with more confidence when more material is available. If the amount of material is marginal or insufficient, only partial sequence information can be obtained. One problem frequently encountered is N-terminal blockage of proteins/peptides. When the N-terminal amino group is modified, in most cases with an acyl group, the Edman degradation cannot proceed. In these cases, internal sequence analysis after proteolytic cleavage and isolation of fragments can be used to obtain sequence information.
Internal sequence analysis of proteins after SDS-PAGE separation
A powerful procedure for the identification of the partial amino acid sequence of proteins capitalizes on the high resolution afforded by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The protein can either be excised from the gel or from the membrane after electro-blotting. The protein isolated in this way is then treated with proteolytic enzyme, most commonly trypsin, and the resulting fragments are separated by reversed phase HPLC. These peptides can then be analyzed and the sequences can be used in database searches to determine identity or homology to known proteins or, in the case of unknown sequences, to design oligonucleotide probes/primers for screening purposes.
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