Akta explorer 100 fplc system

Moth cytochrome C 88–103 peptide (MCC_88–103; abbreviated as MCC) and previously characterized variants (12 (link), 78 ) were synthesized and lyophilized on campus (D. King, Howard Hughes Medical Institute Mass Spectrometry Laboratory at University of California, Berkeley) or commercially (Elim Biopharmaceuticals, Hayward, CA). A short flexible linker of three amino acids and terminal cysteine was added to the C terminus for fluorophore labeling. The sequences are as follows: MCC (ANERADLIAYLKQATK), MCC(C) (ANERADLIAYLKQATKGGSC), K3 (ANERADLIAYPKAATKF), K3(C) (ANERADLIAYPK-AATKFGGSC), T102S (ANERADLIAYLKQASK), T102S(C) (ANERA-DLIAYLKQASKGGSC), and Null(C) (ANERAELIAYLTQAAKGGSC).
For dye conjugation, the cysteine-containing peptide sequences were reacted with the maleimide-containing organic fluorophore of interest (Atto647N, Atto565, or Atto488; Atto-Tec GmbH, Siegen, Germany) in phosphate-buffered saline (PBS) with a trace amount of 1-propanol, as previously described (9 ). The labeled peptides were purified using a H₂O/acetonitrile gradient on a C₁₈ reverse-phase column (Grace Vydac, Deerfield, IL) in the AKTA Explorer 100 FPLC system (Amersham Pharmacia Biotech, Piscataway, NJ). Mass spectrometry was used to confirm the peptide identity after purification.