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Fluorescence‐labeled peptides as isoelectric point (p I ) markers in capillary isoelectric focusing with fluorescence detection
Author(s) -
Shimura Kiyohito,
Kasai KenIchi
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501601245
Subject(s) - isoelectric focusing , isoelectric point , fluorescence , chromatography , chemistry , capillary electrophoresis , detection limit , laser induced fluorescence , biochemistry , physics , quantum mechanics , enzyme
Commercially available peptides, mostly angiotensin derivatives, were labeled at their N ‐terminal amino group with 5‐carboxytetramethylrhodamine succinimidyl ester, to obtain fluorescent p I markers for capillary isoelectric focusing with fluorescence detection. The labeled peptides were purified by reversedphase chromatography. They were well separated on isoelectric focusing in a polyacrylamide gel slab and their p I s were determined by comigration with protein‐pI markers. The fluorescent markers could be detected as sharp peaks in capillary isoelectric focusing with laser‐induced fluorescence detection (He‐Ne laser, 1 mW, 543.5 nm). The detection limit was found to be around 3 × 10 −12 M (0.8 amol). Tetramethylrhodamine‐labeled pea lectin (3 pg) was subjected to capillary isoelectric focusing and the p I s of the fluorescent derivatives of the lectin were determined by using the fluorescence‐labeled peptides as p I markers.