A PREPARATIVE METHOD FOR SEQUENCING PROTEINS AND PEPTIDES - IN-SITU GEL STAINING WITH SUBSEQUENT PASSIVE ELUTION ONTO POLYVINYLIDINE DIFLUORIDE MEMBRANES

A preparative method for obtaining both N-terminal and internal peptid e amino acid sequences from purified proteins is reported. The methodo logy reliably yields high fidelity signal from between 14 to 30 residu es per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of prot eins during preparative sodium dodecyl sulfate-polyacrylamide gel elec trophoresis (SDS-PAGE), and the utilisation of microconcentrators to r epeatedly concentrate small amounts of proteins onto a small polyvinyl idene difluoride (PVDF) disc until sufficient amounts have been adsorb ed so as to give a strong sequencing signal. The protein elution and s ubsequent adsorption can be monitored visually with a dye and the fina l product, a PVDF disc with the adsorbed protein or peptide, can be di rectly inserted into the automated amino acid sequencer.