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Radioiodination of protein

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Radioiodination (by Jun Takagi,6/16/2000) Purpose and backgrounds *Principle of radioiodination* Addition of oxidizing reagents (such as chloramine-T or peroxidase+H₂ 0₂ ) converts I^- to I+ or I^3- . This highly reactive molecule attacks o-position of tyrosine (or in some case, histidine is also labeled). About nuclide... 125-I decay: electron capture (g-ray, 0.035 MeV) half life: 60 days requires >3 mm lead plate to block >90% emission. Glass, plastic, or water cannot block. Chemical properties... Usually shipped as Na¹²⁵ I in alkaline solution at 1 mCi(37 MBq)/10µl. Easily evaporates when converted into molecular gas (I2) form. Therefore, you MUST NOT freeze the vial. Upon freezing, I₂ will sublimate. Always stabilize ionized form (I^- ) and avoid low pH and oxidizing condition for waste solution containing inorganic I^- . To accomplish this, add 1/10 vol of stabilizer (see below) to the waste solution. Handling of Na¹²⁵ I soln in v-vial* IODINE-125(Amersham; IMS30) will be shipped in v-vial with rubber septum. NEVER remove the septum! The solution must be taken out from the vial using Hamilton microsyringe with sharp needle.

				Materials For procedure 1 • 0.1 M phosphate buffer, pH 7.0 • Tris-buffered saline (TBS) • 5% BSA in TBS • stabilizer soln (10% sodium thiosulfate+0.1N NaOH) • IODO-BEAD™(PIERCE) • Hamilton microsyringe (model 702, 25µl, needle gauge 22S, point style #2) • desalting column (Bio-rad DG-10 etc.) • column stand For procedure 2 • PBS containing 20 mM glucose • Hepes-Tyrode buffer (or any buffer compatible with your subsequent experiments with labeled cells) • lactoperoxidase (Sigma L-8257) dissolved in PBS at 1 mg/ml • glucose oxidase (Sigma G-7016) dissolved in PBS at 5 U/ml Procedure i) Radioiodination of protein in solution *Reading a free booklet available from Amersham ("Guide to radioiodination techniques") is recommended. 1. In clear plastic tube, add 200µl of 0.1M phosphate buffer, pH 7 2. Add 5µl (500µCi) of 125-I using microsyringe 3. Add 1-2 pieces of IODO-BEADs^¶ 4. incubate at room temp, 5 min 5. Add your protein(1-100µg, 25 µg is good for IgGs or Fab) 6. Incubateat room temp, 10-25 min 7. Take reaction mixture (leave beads) and apply on a desalting column ^§ 8. Collect fractions (0.5 - 1 ml/fr.) ^¶ Prior to use, washed three times with phosphate buffer to remove debris on the beads) ^§ Block nonspecific binding site by applying 1ml of 5% BSA-TBS followed by equilibration with TBS.

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