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Sequence Tag

Automated Edman degradation, which produces N-terminal protein sequence, is a common identification method for PVDF-bound proteins. Sequencing is usually done for 15 cycles, and identity is established by matching the sequence obtained against those in protein databases. Internal protein sequencing is also possible, but is a more labor-intensive procedure.

In our early work with 2-D PAGE reference maps we almost exclusively used N-terminal sequence analysis for protein identification. However, with a throughput of one protein per sequencer per day, this approach was too slow for the identification of large numbers of protein samples. To address this problem but continue to use the specificity of protein sequence data, we now use a combination of Edman degradation and Amino acid analysis techniques for rapid protein identification. PVDF-bound proteins are sequenced for only three or four cycles at the amino terminus, to create a N-terminal sequence tag. We use fast but low repetitive yield sequencing programs as only a few residues of sequence are required. After sequencing, the same PVDF-bound protein sample which was sequenced is used for AA analysis. Protein identification is then achieved by matching AA compositional data, estimated protein pI and Mw, and the protein "sequence tag" against the Swiss-Prot database (see AACompIdent tool for details). This approach provides a powerful and unambiguous identification method which, as compared to extensive N-terminal protein sequencing, increases sample throughput five to ten fold and greatly reduces reagent costs.



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