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.
The protein is immobilised on a PVDF membrane or TFA-treated filter. 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.