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Radioiodination of protein in solution
Posted on Friday, October 17, 2003
Addition of oxidizing reagents (such as chloramine-T or peroxidase+H202) converts I- to I+ or I3-. This highly reactive molecule attacks o-position of tyrosine (or in some case, histidine is also labeled).
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.
Labeling efficiency (incorporation) should be 10-90% (depends on amount of protein added). Collect appropriate fraction (in this case, fr #3+4), add 1/4 vol of 5%BSA*, and store either at 4°C or -80°C in aliquots.
*addition of high conc. BSA will guard your dilute protein from decomposition by radiation, but should not added if it interfere with subsequent experiments. However, labeled and dilute protein without carrier protein tends to flocculate and easily loses its activity.
• 0.1 M phosphate buffer, pH 7.0
• Tris-buffered saline (TBS)
• 5% BSA in TBS
• stabilizer soln (10% sodium thiosulfate+0.1N NaOH)
• Hamilton microsyringe (model 702, 25µl, needle gauge 22S, point style #2)
• desalting column (Bio-rad DG-10 etc.)
• column stand
Submitted by: manny440