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Edman Sequencing

N-terminal and internal protein sequencing are achieved by sequential Edman reactions. In this process, the N-terminal amino acid is reacted with PITC (phenylisothiocyanate) to form a phenylthiocarbamyl (PTC) protein. The PTC protein is then cleaved with trifluoroacetic acid, resulting in the formation of an intermediate anilinothiazolinone (ATZ). The intermediate is converted to the more stable phenylthiohydantoin (PTH) amino acid derivative and subsequently separated by HPLC, compared against a standard, and identified by the sequencer software.

Protein sequence analysis is performed in our facility using an ABI 492 Procise cLC sequencer, with routine analysis in the low picomole range. We need 10 picomoles (500 ng of a 50 KDa protein) of pure, salt free protein, although sequencing runs of 20 or more residues have been obtained with samples as small as 1 picomole. In general, the more sample that can be submitted, the better the quality and quantity of the data obtained. Similarly, successful sequencing depends upon the purity of the sample, volume and buffer. Protein should be at least 80% pure. Please contact the facility operating manager for further information on core services of this nature.

The protein can be submitted in soluble form resolved by HPLC or as a SDS-PAGE-resolved band transferred onto PVDF membrane and stained with Coomassie blue or Ponceau Red. See our sample protocols for more detailed information.

Blocked N-termini provide the single largest impediment to protein sequence analysis. An estimated 50-80% of all proteins naturally have chemically modified N-termini. Depending upon the nature of the modification, some N-terminal blocking groups can be removed with mild acid treatment. Failing this "quick fix" the standard solution is to carry out chemical or enzymatic cleavages followed by HPLC purification.

Many proteins may be blocked at the N-terminus by modification during manipulation and purification. Modifications include: formyl groups, acetyl groups, and pyroglutamic acid residues. Modification to the N-terminus may result from the following manipulations: handling steps at elevated pH (>9.0), using inferior grade reagents and water (use the highest quality available), exposure to elevated temperatures (if glutamine is the N-terminal residue), exposure to protease inhibitors (some inhibitors may react with amino groups), use of formic acid (e.g. in CNBr cleavage) may formylate the N-terminus (use 50-70% TFA as an alternative), and exposure to urea that has not been deionized on an ion exchange resin (e.g. Amberlite) prior to use

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