Iodogen

The best radiolabeling technique for SASD is to use the Iodogen method (Shephard et al., 1988) described in Chapter 12, Section 3. The following suggested protocol for using SASD was based on the method described in the Thermo Fisher Catalog. 

Specific radioactivity of 1 X 105 cpm of 125I per microgram of protein easily can be obtained using Iodogen. Iodination efficiencies are typically 60 percent or better and may be controlled by regulating the amount of I- concentration added to the reaction. 

The following protocol describes the use of Iodogen for the radioiodination of proteins and peptides. 

In a fume hood, dissolve 10-100 pig of Iodogen (Thermo Fisher) in 100-500 pil of chloroform, methylene chloride, or DMSO. The use of 10 pig of Iodogen per 100 pig of protein or 107 cells to be iodinated will result in good incorporation yields. 

Rinse the plated reaction vessel once with sample buffer to remove any loose particles of Iodogen 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 Iodogen particles break off from the sides of the vessel. To assure safe handling, carrier Nal may be added to the reaction mixture to a final concentration of 1 mM. 

Salacinski, P. R. P, McLean, C., Sykes, J E. C, Clement-Jones, V. V, and Lowry, P. J (1981) Iodmation of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, l,3,4,6,-tetrachloro-3a,6a-diphenyl glycolunl (iodogen). Anal. Biochem. 117, 136—146. 

Iodogen is available from Pierce (Rockford, IL). This can now be obtained precoated on tubes. 

I]-Diphtheria toxin ( 4.5 pCi/pg) is produced by reaction with Na[125I] using the iodogen method (36). 

Antimyosin Fab was labeled with 125I and 123I by the Iodogen iodination method (61) because of its gentler nature of compared to chloramine T. Free and AM-bound radioactivity were separated as described above. 

Use iodogen-coated tubes for radioiodination of streptavidin and b-catalase. The iodo-beads technique, described in the recommendations of the manufacturer (Pierce) is also suitable and offers similar results. 

Dissolve 1 mg of iodogen in 1 mL of anhydrous chloroform, add 100 pL of this solution to a 5-mL glass tube, and evaporate chloroform under nitrogen gas. 

Albumin was radiolabeled in the following manner. Defatted human serum albumin, 10 mg, was dissolved in 1 mL of PBS. From this solution, 100 jjlL was placed in an Iodogen-coated reaction vessel (prepared by P. Kulkarni). A 25- xL aliquot of 0.25M phosphate buffer solution (pH 7.52) was added, and the vessel was chilled over ice for 10 min. A 5-jxl aliquot of Na125I, 1-1.5 mCi, was then added, and the vessel was rotated slowly several times. The vessel was again placed in an ice bath for 2 min and then incubated for an additional 15 min at room temperature. At the end of the incubation period, 100 xL of PBS was added to the reaction vessel, and the solution... 

Figure 76.3 iodogen. This reagent is a miider aiternative to chioramine T. 

Peroxidases Enzymatic labeling is a milder alternative to chemical oxidants. It can be accomplished without deactivation or degradation. It is thus suitable to label very sensitive molecules, such as biomolecules. The labeling yields are close to those obtained with chloramine-T and iodogen, and no carrier-added radioiodination can be obtained (Davidson, 1987). 

During the iodination with the oxidizer chloramine T, all reactants are present in solution (one-phase system). Pierce offers oxidizers that were applied to a solid phase (two-phase system iodobeads, iodogen). lodobeads are N-chlorobenzene sulfonamides attached to polystyrene beads. Iodogen is a hydrophobic chloramine T derivative applied to the wall of the reaction vessel. After the reaction with iodobeads and iodogen, the solid phase with the oxidizer can easily be separated from the reaction mixture. Hence, the addition of reducing agent (bisulfite) is unnecessary, which spares the sensitive disulfide bridges of some proteins. In addition, N-chlorobenzene sulfonamide is a milder oxidizer than chloramine T. 

Pierce iodination-labeling tubes (Thermo Fisher Scientific), glass tubes coated with iodogen reagent, the labefing reaction takes place at the surface of the water-insoluble oxidant. 

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