Protein Protocols Protein Protocols

Radioiodination Using IODO-GEN 975
tion (1600g for 30 min at 4°C). Under these conditions, >90% of the radioactivity is
bound to antibody.
4. Notes
1. A systematic comparison of the properties of different IODO-GEN-coated surfaces has
concluded that soda-lime glass produced the most rapid rate of oxidation of Na 125I (10).
However, irreversible binding of most peptides to the walls of polypropylene tubes is
much less than to glass and so clear polypropylene Eppendorf tubes are routinely used in
the author’s laboratory. The use of borosilicate glass tubes (10) and polysytrene tubes is
not recommended. Glass test tubes (12 mm × 75 mm) precoated with IODO-GEN (50 µg)
are available from Pierce.
2. The choice of column is dictated by the nature of the radiolabeled peptide to be purified.
For relatively small (Mr < 3000) peptides, good resolution and recoveries are generally
obtained with (0.46 × 25 cm) narrow pore (80 Å), 5-µm particle size octadecylsilane (C18) columns such as Supelcosil LC-18-DB (Supelco, Bellefonte, PA), Ultrasphere ODS
(Beckman, Duarte, CA), or Spheri-5 RP-18 (Brownlee/Applied Biosystems, Foster City,
CA). For purification of radiolabeled tracers of higher molecular mass (Mr > 3000), the
use of columns containing wide-pore (300 Å) 5-µm particle size C18 packing materials is
recommended. Suitable columns include Vydac 218TP54 (Separations Group, Hesperia,
CA), Spherisorb wide-pore C18 (Phase Separations; U. K.), Waters Delta-Pak C18 (Millipore, Milford, MA), and Ultrapore C18 (Beckman). For purification of tracers of
molecular mass >10,000, such as the pituitary glycoprotein hormones, sharper peaks and
better recoveries of radioactivity may be obtained using wide-pore silica with propyl and
butyl carbon loading. Suitable columns include Beckman Ultrapore C3 and Vydac
214TP54 C4 columns.
3. The reaction is not markedly pH sensitive and high incorporations of 125I may be achieved
in the pH range 6–9 (11).
4. Suitable water can be obtained using a Milli-Q purification system (Millipore) supplied
with water that has been partially purified by single distillation or by treatment with a
deionization resin.
5. A low reaction temperature is important in minimizing damage to the radiolabeled
peptide/protein. For example, the use of IODO-GEN at room temperature to prepare
125I-labeled human growth hormone resulted in the production of tracer containing a significant
amount of the hormone in an aggregated form whereas only the radiolabeled
monomer was produced when the reaction was carried out at 0°C (12).
6. As many peptides are relatively insoluble in buffers of neutral pH, it is recommended that
the peptide first be dissolved in a minimum volume (approx 5 µL) of 0.1% (v/v) trifluoroacetic
acid/water and the volume made up to 100 µL with 0.2 M sodium phosphate
buffer, pH 7.5.
7. The optimum reaction time must be determined for each peptide and protein, but some
general guidelines can be given. For small peptides (