Fluorescent Conjugate Formation Protocol

Fluorescent conjugates were constructed using Alexa Fluor 488 5-TFP (Life Technologies, Eugene, OR, USA) following the manufacturers protocol and methodology previously described in [15 (link)]. Saporin SO6 or OKT10-SAPORIN were added to 100 µL carbonate buffer (1 M NaHCO₃, pH 9.0) and 100 µL of Alexa Fluor 488 5-TFP (10 mg/mL in DMSO) and stirred for one hour at room temperature. Unconjugated fluorophore was removed by dialysis against PBS at 4 °C. The Beer–Lambert law was used to determine the concentrations of the fluorescent conjugates by measuring the absorbance of the samples at 280 and 495 nm using a Hitachi U1100 Spectrophotometer.