What method is used to do the sequencing?
The process uses the very well established technique of Edman degradation. Here, one amino acid at a time is removed from the N-terminal of the protein by alternately reacting the protein with the Edman reagent, (phenyl isothiocyanate, PITC) and cleaving the resulting compound off the protein with acid. Identification of the amino acids is achieved by their elution times on HPLC compared to a standard mixture. The protein is immobilised inside the instrument by either blotting it first onto a PVDF membrane or by its adsorption onto a biobrene treated glass fibre filter. The cleaved amino acids are converted to their PTH derivatives, (phenylthiohydantoin) before being automatically injected onto the onboard HPLC instrument. Data from the HPLC is collected on a computer for visual calling of the sequence. Back to top.
