Perfectsil 300 ods c18

Peptides were synthesized on solid phase by standard Fmoc chemistry. For cysteine labeling, the C4 peptide (RRYCKSTEL) was incubated with 5-iodoacetamide fluorescein (Sigma-Aldrich) or Alexa Fluor 647 maleimide (Thermo Fisher) in PBS DMF buffer (8.1 mM Na₂HPO₄ pH 6.5, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 20% (v/v) DMF) for 1 hr at 20 °C using a molar fluorophore excess of 1.2. Samples were purified by reversed-phase HPLC (Jasco; PerfectSil 300 ODS C₁₈) applying a linear acetonitrile gradient from 5–80% supplemented with 0.1% (v/v) TFA. Purified peptides were snap frozen in liquid nitrogen and lyophilized (Lyovac GT2, Heraeus).

C4 peptide (RRYCKSTEL) was synthesized on solid phase using standard Fmoc chemistry. For fluorophore labeling, C4 peptide was incubated with a 1.2 molar excess of 5-iodoacetamide-fluorescein (Sigma-Aldrich) or ATTO655-maleimide (ATTO-TEC) in PBS DMF buffer (8.1 mM Na₂HPO₄ pH 6.5, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH₂PO₄, 20% [v/v] DMF) for 1 hr at 20°C. Peptides were purified by reversed-phase HPLC (Jasco; PerfectSil 300 ODS C₁₈), utilizing a linear acetonitrile gradient from 5% to 80% supplemented with 0.1% (v/v) TFA. Purified fluorescently labeled peptides were snap-frozen in liquid nitrogen and freeze-dried (Lyovac GT2, Heraeus).