1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (Chloroglycoluril), also known as Iodo-Gen®, is one of the best reagents used for iodination of proteins, hormones, antibodies, viruses, cell membranes, etc. This method was first developed by Fraker et al. This method of iodination with Iodo-Gen® is simple and inexpensive. Iodo-Gen® is water-insoluble and hence used in solid-phase iodination. Iodo-Gen® is used to incorporate radioactive iodine in aromatic amino acids like tyrosine. It iodinates proteins in oxidizing conditions, so there is no need for using excessive reducing agents that affects protein stability. The proteins are not directly subjected to oxidation in this method as the reagent sticks to the wall of the tube and protein is in solution. Either Iodo-Gen® is coated on the walls of glass vials or on polystyrene beads. Iodo-Gen® is dissolved in an organic solvent and the organic solvent is removed from the vial by using nitrogen. The Iodo-Gen® that sticks to the wall of the vial or beads later iodinates proteins in solution. Protein can be separated easily from unreacted reagent as Iodo-Gen® is water-insoluble.
Reagents required:
- Iodo-Gen® from G-Biosciences
- Chloroform or dichloromethane (DCM)
- Sodium iodide (125 I)-500 µCi (per 100 µg protein)
- Buffer 1: 50mM Phosphate buffer, pH 7.5
- Buffer 2: 50mM Phosphate buffer, 0.15 M NaCl, 1 mM KI, pH 7.5
- Buffer 3: 50mM Phosphate buffer, 0.2% BSA. 0.01% Sodium Azide, pH 7.5
- Protein - 5-10 µg
Procedure:
Preparation of Iodo-Gen® tubes
5 mg Iodo-Gen® was dissolved in chloroform or dichloromethane (DCM) to a concentration of 0.1 mg/ml. 20-30 µl of this solution is added to a clean glass tube or polypropylene tube and dried under Nitrogen gas at room temperature. This step is to be performed slowly to avoid formation of clumps of Iodo-Gen® on the walls. The dried tubes were closed and stored at 4°C (1 month) or at -20°C (3 months) until further use. A large number of tubes can be coated with Iodo-Gen® in bulk and stored for further use because of the high stability of Iodo-Gen® coated tubes.
Iodination of Proteins with Iodogen
Direct method:5-10 µg of Protein was dissolved in 20 µl of phosphate buffer (Buffer 1) and 5 µl of Sodium iodide, added to Iodo-Gen® treated tubes and mixed gently for 30-45 seconds. Then the solution is transferred to a fresh tube with Phosphate buffer with KI, 0.15 M NaCl solution (Buffer 2). An extra step of 5-minute incubation of sample before gel filtration helps in conversion of unincorporated iodine to molecular iodine and thus avoids iodination of BSA in column buffer in the next step.
Indirect method using crosslinker
A stock solution of NHS-ester crosslinker was prepared by dissolving 5.5 µmol of NHS-ester crosslinker in 50 µl of DMSO. The working solution was prepared by dissolving stock solution in 100 mM Sodium Phosphate buffer pH 7.5 to get a final concentration of 55 nmol. 100 µl of this solution is added to Iodo-Gen® treated vial along with 40 µCi Sodium iodide, 18.5 nmol KI and incubated for 30 seconds. The iodinated crosslinker is then mixed with a protein solution in a separate vial and incubated for 20-30 minutes. Excess iodine and crosslinker are separated from protein by dialysis or gel filtration method.
The procedure of iodination with Iodo-Gen® can be modified depending on protein to be iodinated, reaction time, amount of Iodo-Gen® used and pH of reaction buffer. Note that Increase of Iodo-Gen® incubation time from 30 seconds to 20 minutes show no improvement or decrease of iodination. Besides, shorter times help in avoiding exposure of the protein to harsh external conditions. In iodination reaction, the molar ratio of radioactive iodide per protein is 1-1.2 and the molar ratio of Iodo-Gen® to protein is 8. Iodination with Iodo-Gen® can be performed within a pH range of 6.0-8.5, varies with protein to be labeled. Depending on the protein sample, Sodium phosphate buffer, HEPES, Borate buffer or PBS can be used.
Purification of iodinated Proteins:
The iodinated proteins are then purified either by dialysis or by using gel filtration column, such as G-Biosciences SpinOUT™ GT-600 or SpinOUT ™ G-Acryl 600 columns, using column buffer (Buffer 3). 400 µl of fractions were collected and protein containing fractions were further checked for radioactivity by RIA or Isoelectric focusing.
References:
- Fraker, P. J. and Speck, J. C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloramide 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril. Biophys. Res. Comm.280, 849–857.
- Markwell MA, Fox CF.Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril, Biochemistry1978 Oct 31;17(22):4807-17.
