Protein Protocols Protein Protocols

Radioiodination Using IODO-GEN 973
2.2. Chemicals
2.2.1. Iodination Reagents
1. IODO-GEN (Pierce, Rockford, IL): The reagent should be stored in a desiccator in the
freezer and the bottle protected from light.
2. Dichloromethane (stabilized, HPLC grade): Redistillation is not required.
3. Carrier-free Na + 125 I – in 0.1 M NaOH (3.7 GBq/mL; 100 mCi/mL, Amersham Pharmacia
Biotech, Piscataway, NJ).
4. 0.2 M Disodium hydrogen phosphate–sodium dihydrogen phosphate buffer, pH 7.5 (see
Note 3).
2.2.2. Chromatography
1. Solvent A: Add 1 mL of trifluoroacetic acid (Pierce HPLC/Spectro grade) to 1000 mL of
water (see Note 4).
2. Solvent B: Mix 700 mL of acetonitrile (Fisher optima grade) with 300 mL of water and
add 1 mL of trifluoroacetic acid. The solvents should be degassed, preferably by sparging
with helium for 1 min, but passage through a filter is unnecessary.
3. Method
3. 1. Radioiodination
1. Dissolve 1.5 mg of IODO-GEN in 2 mL of dichloromethane.
2. Pipet 20 µL of the solution into a polypropylene tube and remove the solvent in a gentle
stream of nitrogen or argon at room temperature. The aim is to produce a film of IODO-
GEN on the wall of the tube. If the reagent has formed a visible clump, the tube should be
discarded and a new tube prepared. According to the manufacturer’s instructions (Pierce),
the tubes can be stored in a vacuum desiccator for up to 2 mo, but it is recommended that
a tube is prepared freshly for each reaction. The tube is set in an ice bath for 10 min prior
to the iodination (see Note 5).
3. Dissolve 10 nmol of the peptide (see Note 6) in 0.2 M sodium phosphate buffer, pH 7.5
(100 µL) and pipet the solution into the chilled IODO-GEN-coated tube.
4. Add the Na + 125I – solution (5 µL; 0.5 mCi or 10 µL; 1 mCi depending on the quantity of
tracer required).
5. Allow the reaction to proceed for between 1 and 20 min (see Note 7). The contents of the
tube should be gently agitated by periodically tapping the side of the tube with a gloved finger.
6. Reaction is stopped by aspirating the contents of the tube into a solution of 0.1% (v/v)
trifluoroacetic acid–water (500 µL) contained in a second polypropylene tube. The reaction
vessel may be washed with a further 200 µL of trifluoroacetic acid–water and the
washings combined with the reaction mixture.
3.2. Chromatography
1. Prior to carrying out the iodination, the column is equilibrated with 100% solvent A at a
flow rate of 1.5 mL/min for at least 30 min. In the case of more hydrophobic peptides/
proteins, the column is equilibrated with starting solvent containing up to 30% solvent B
(21% acetonitrile).
2. The instrument is programmed to increase the proportion of solvent B from starting conditions
to 70% (49 % acetonitrile) over 60 min using a linear gradient (see Note 8).