Materials

• Protein at 1 mg/ml in BSB pH 8.6

• ¹²⁵I 1 mCi/ml

• ICl at 1 mg/ml in 0.1 N HCI

(To make 1 mg/ml ICl from dense ICl - add 0.1 ml dense ICl to 330 ml 0.1 N HCl with rapid mixing. Aliquot in glass vials and store -20C.)

Methods

1. Tube A - 0.5 ml protein in BSB (0.5mg)
2. Tube B - 0.5 ml BSB ( + 5 µl 0.1 N NaOH to neutralize HCl from ICl); add 50 µl ICl; mix
3. Quickly add 2 drops ¹²⁵I to Tube B and add contents of tube B to tube A.
4. Mix and immediately transfer entire reaction to dialysis tubing.
5. Dialyse vs 500 ml BSB; change dialysis 2X; the first 2 dialysis should also be 0.02 M KI

This will yield approximately 2 or 3 atoms Iodine per molecule protein (IgG) according to the following formula:

(formula wt. of ICl - 163)

McFarlane, A.S. Nature, 182, 53 (1958)

©1995-2009 Loyola University Chicago Stritch School of Medicine.
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