Protein Sequencing (Edman Degradation)

The protein is reacted with the Edman reagent (phenylisothiocyanate, PITC) which modifies the N-terminus and the side-chains of Lys residues. Under controlled conditions, the N-terminal residue is released and converted to the phenylthiohydantoin (PTH) derivative which is identified by HPLC. Successive cycles of chemistry provide the N-terminal sequence of the protein. Some eukaryotic proteins are blocked at the N-terminus by acetylation and are therefore unable to be sequenced directly by this method. N-termini may also become blocked by chemical reactions such as carbamylation.

Protein from reverse-phase HPLC or electroblotted onto PVDF. We recommend blocking the protein's thiols with iodoacetic acid if Cys is to be detected directly. The appropriate amount of protein is 2 to 100 pmol.

Typically, we bind the protein to a PVDF membrane or TFA-treated filter to immobilise it. Sequencing is then performed automatically for the agreed number of cycles.

Applied Biosystems Procise Sequencer (model 492) attached to a model 140C HPLC system with a model 785A absorbance detector operated under the control of 610A software.

The primary sequence is deduced automatically and validated manually.

Chromatograms from each cycle and a report are provided, describing the analysis undertaken and the deduced sequence(s). The report is either provided as hardcopy or sent by email, as Word document.
Raw data are in a proprietary format and are not included with the report. Data are archived by the APC.
If the data are used as part of a scientific publication, due reference to the APC should be made.