Radioiodination and Purification of Proteins

Briefly, Fcp5, p5, Fc2a, and mAb 11-1F4 were radioiodinated with 2 mCi iodine-125 (¹²⁵I; Perkin Elmer) in the presence of 10 µg of chloramine T. The radiolabeled products were purified by size-exclusion gel filtration using either; Sephadex G-25 (PD10; GE Healthcare, Pittsburgh, PA, USA), Aca44 (Sigma-Aldrich), or Aca34 (Sigma-Aldrich) with a mobile phase of PBS containing 0.1% (w/v) gelatin, as previously described (29 (link)). The radiochemical yield was estimated by measuring the amount of ¹²⁵I recovered in the purified pooled product relative to the amount of added ¹²⁵I. The radiochemical purity and integrity of the purified product was assessed by SDS gel electrophoresis using 10% polyacrylamide gels followed by phosphor imaging (Cyclone Storage Phosphor System, Perkin Elmer, Shelton, CT, USA).