Protein/Peptide Amino Acid Sequencing

Two instruments are currently used in the facility: the 477A protein/peptide sequencing system with an in line PTH-analyzer from Applied Biosystems and a Porton Instrument with an in line PTH analyzer from Beckman Coulter.

Method

Protein/peptide sequencing is performed by the Edman chemistry and involves the removal of one amino acid each cycle from the N-terminus of the peptide. This is a cyclical process, which is repeated roughly each hour. The steps involved are: 1) coupling of PITC to the free N-terminus of the protein (around 80% of eukaryotic proteins are N-terminally blocked and do not undergo the coupling step. [Blocked proteins cannot be sequenced as such. Internal peptide fragments need to be prepared and purified before sequencing can be performed]; 2) cleavage of the N-terminal amino acid. 3) conversion of the cleaved amino acid to the stable PTH-amino acid which is then analyzed by HPLC reversed phase separation. Each step is performed under a well-defined set of conditions.

Sample Preparation and size

At least ten picomoles of protein is needed for sequencing. Gel-purified samples should be electroblotted to PVDF membrane and stained with coomassie blue. Submit the entire blot with the band of interest marked. Alternatively, samples containing at least 10 picomoles of pure peptide/protein can be supplied to the facility either as a dry sample or in a small volume of water that can either be applied directly to the sequencer (about 30 µl) or in a bigger volume that can be dried in a speed vac in a single tube. Non-amine buffers are tolerated by the sequencer in the 1-5 mM range; amine buffers such as Tris are not compatible with th Edman reactions. Under some conditions, smaller amounts of sample can be successfully sequenced. contact us beforehand for small samples and alternative methods of sample submission.

Data Provided

Copies of the data sheet and the request form. The chromatograms will be stored at the facility. Copies of the chromatograms will be supplied to investigators upon request.

Turn-Around Time

For a single sequence the turn-around time is 2-3 working days. Additional sequences will take 1-2 additional days per sample.

Fee Schedule

	MCW	Others
protein sequencing	$200	$400
SDS-PAGE, electroblotting, and sequencing	$300	$500
2-D electrophoresis, electroblotting, and sequencing	$350	$550