2-DIMENSIONAL ELECTROPHORESIS AS A COMPLEMENTARY METHOD OF ISOLATING PEPTIDE-FRAGMENTS OF CLEAVED PROTEINS FOR INTERNAL SEQUENCING

To determine the primary structure of proteins, usually proteolytic en zyme digests are separated by reversed-phase high-performance liquid c hromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the en tire sequence. However, there are many N-terminal-blocked proteins and /or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successful ly isolated by the use of two-dimensional electrophoresis, after diges tion. Electroblotted spots were easily sequenced to identify those dif ficult fragments which could not be obtained using HPLC.