Protein Protocols Protein Protocols

974 Conlon
Fig. 2. RP-HPLC on a (0.46 × 25 cm) Vydac 218TP54 (C 18) column of the reaction mixture
following incubation of 10 nmol of rat galanin with 0.5 mCi of Na 125 I in an IODOGEN-coated
tube for 2 min at 0°C. Fractions (1 min) were collected and the fraction denoted by the bar
contained tracer of high specific activity (74 TBq/mmol). The dashed line shows the concentration
of acetonitrile in the eluting solvent.
3. The reaction mixture is injected onto the column (see Note 9). The fraction collector,
programmed to collect 1-min fractions, is started and the linear elution gradient is begun.
A total of 60 fractions are collected.
4. At the end of the chromatography, the column is irrigated with 100% solvent B for 30 min.
The column may be stored in this solvent.
5. The radioactivity in aliquots (2 mL) of each fraction is counted in a gamma scintillation
counter (see Note 10 for optimum storage conditions of tracer).
The results of a typical radioiodination are illustrated in Fig. 2. The reaction mixture
comprised 10 nmol of rat galanin and 0.5 mCi of Na + 125 I – and the reaction time was
2 min. Unreacted free iodide was eluted at the void volume of the column (fractions 5
and 6). The fraction denoted by the bar (tube 51) was of high specific activity (approx
74 TBq/mmol; 2000 Ci/mmol). The earlier eluting minor peak of radioactivity (tube 49)
probably represented the diiodotyrosyl derivative. Before use in radioimmunoassay or
radioreceptor studies, the quality of the tracer is assessed by incubating an aliquot
(approx 20,000 cpm) with an excess of an antiserum raised against galanin (11000
dilution) in 0.1 M sodium phosphate buffer, pH 7.4 (final volume 300 µL) for 16 h at
4°C. Free and bound radioactivity are separated by addition of 100 µL of a 10 mg/mL
solution of bovine γ-globulin (Sigma, St. Louis, MO) and 1 mL of 20% (w/v) solution
of polyethylene glycol 6000 (Sigma, approx M r 8000) in water followed by centrifuga-