Chloramine-T Protocol

Na I solution, 3.7 GBq/ml 0.25 M sodium phosphate buffer, pH 7.5 50 mM sodium phosphate buffer, pH 7.5 5 mg/ml chloramine-T (sodium N-chloro-p-toluenesulfonam-ide) in Soln. C 

Prepare Soln. D, E, and G immediately before starting the experiment. 

Pipet 5-10 pi of Soln. A (4-7 MBq) into a 4-ml polystyrene or siliconized glass test tube. Then add 25 pi Soln. B, followed by 10 pi of protein solution (2-5 pg, dissolved in Soln. C), 10 pi Soln. D, and 100 pi Soln. E. Vortex after each addition. Fill up to 1 ml with Soln. G and count for total radioactivity. 

Equilibrate a 1 x 10 cm Sephadex G-25 column with 50 ml Soln. F. Apply the iodinated sample, elute with Soln. F, and collect 1-ml fractions. The iodinated protein appears in the void volume, and free iodine-125 elutes with the total volume. Estimate its radioactivity and calculate the specific radioactivity (Bequerel per milligram of protein). 

If the iodinated protein will be used in bioassays, check for biologic activity (antigenicity, enzymic activity, ligand binding, etc.) in comparison with the not-labeled starting material. 

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