Protein sequencing from N-terminus

  Reagent

  Phenylisothiocyanate (PITC)

  Cycle

  1. Coupling pH = 8 - 8.5

  2. Cleavage of peptide bond (no water!, acidic)

3.Rearrangement of ATZ to PTC - Aa then to PTH - Aa (aqueous, acidic)

 ( PTH = Phenylthiohydantoine)

  Setup

  automated in sequenator either gas or liq. phase

  Sample

Sample adsorbed on glass fiber disc or PVDF

 (Peptide sticks to disk while being degraded)

  Detection

  HPLC WITH UV - Detection (269 nm), reversed phase

  Sensitivity

  1 - 10 pmol Aa

  Initial Yield

  50 - 80 % of proteins

  Repetitive Yield :

  80 - 96 % / cycle

  Example

  10 pmol protein / 95 % rY = 40 Cycles = 40 Aa

  real

  5 µg = 10 - 15 AS