IODO-GEN

Radiolabel 55 nmol of SASD using IODO-GEN (Thermo Fisher) and 40 pCi Na 125I for 30 seconds. Do not use chloramine-T, since termination of the iodination reaction with this reagent involves addition of a reducing agent which may cleave the disulfide bonds of the crosslinker. 

Figure 12.5 IODO-GEN is a water-insoluble oxidizing agent that can react with 1251 - to form a highly reactive mixed halogen species, 125IC1. This intermediate can add radioactive iodine atoms to tyrosine or histidine side chain rings.

Fig. 17. Chloramine-T (left) and Iodo-gen (right) reagents, used in radio-iodination reactions.

IODO-GEN (l,3,4,6-tetrachloro-3-6-diphenylglycouril) is a better reagent for the lodination of proteins than chloramine-T, is less damaging, and has fewer side reactions than the latter. The insolubility of IODO-GEN in water means that tubes can be precoated with the reagent dissolved in methylene chloride or chloroform. Then the tubes are stored in the dark until required. The reaction is started by adding the protein and radioiodide, and terminated by removing the sample from the reaction vessel. 

The best radiolabeling technique for SASD is to use the IODO-GEN method (Shephard et al., 1988) described in Chapter 8, Section 4.3. The following suggested protocol for using SASD was based on the method described in the Pierce Catalog. 

IODO-GEN (Pierce), first described by Fraker and Speck in 1978, is 1,3,4,6-tetrachloro-3a,6a-diphenylglycouril, an N-haloamine derivative with oxidizing properties similar to those of IODO-BEADS and chloramine-T. The compound is insoluble in aqueous solution, therefore making it a type of solid-phase radioiodination reagent. However, unlike IODO-BEADS, wherein the oxidizing group is immobilized on an-... 

The reaction of IODO-GEN with iodide ion in solution results in oxidation with subsequent formation of a reactive, mixed halogen species, IC1 (Fig. 266). Either 125I or 13 1 can be used in this reaction. The IC1 then rapidly reacts with any sites within target molecules that can undergo electrophilic substitution reactions. Within proteins, any tyrosine and histidine side-chain groups can be modified with iodine within... 

Specific radioactivity of I x 105 cpm of 125I per microgram of protein easily can be obtained using IODO-GEN. Iodination efficiencies are typically 60% or better and may be controlled by regulating the amount of I- concentration added to the reaction. 

The following protocol describes the use of IODO-GEN for the radioiodination of proteins and peptides. 

In a fume hood, dissolve 10—100 xg of IODO-GEN (Pierce) in 100—500 xl of chloroform, methylene chloride, or DMSO. The use of 10 xg of IODO-GEN per 100 xg of protein or 107 cells to be iodinated will result in good incorporation yields. 

Add the IODO-GEN solution to a clean, dry, glass reaction vessel in an amount needed for the quantity of protein to be labeled. Slowly evaporate the solvent in the vessel using a stream of dry nitrogen or other inert gas. Do not use a strong gas jet, since rapid evaporation or turbulence in the solvent solution will cause uneven IODO-GEN distribution with possible clumping. Do not merely leave the vessel to dry in a hood, since contaminants or moisture may get into the... 

Rinse the plated reaction vessel once with sample buffer to remove any loose particles of IODO-GEN that may not be strongly adhered to the surface of the glass. 

Remove the sample from the reaction vessel. This process should terminate the iodination reaction, unless small IODO-GEN particles break off from the sides of the vessel. To ensure safe handling, carrier Nal may be added to the reaction mixture to a final concentration of 1 mM. 

It has been demonstrated that when uracils are iodinated with electrophilic iodine (using chloroamine-T and iodide or Iodo-Gen) in aqueous solution, or NIS in ethanol or chloroform-ethanol, addition products (47 or 48) form initially, eliminating water or ethanol when heated (81RTC267) (Scheme 41). 

Radiolabeling of IT/antibody is accomplished by using the Iodo-Gen method (42), i.e., adding 0.1 mCi 125INa to 50-100 pg protein and removing the free iodine by gel filtration on Sephadex G-25 Microspin column (Pharmacia). 

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