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| 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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| Typical result with Fab fragment labeling (50µg used) | ||||||||||
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. ii).Radioiodination of cell surface proteins 1. Wash cells at least three times with PBS and resuspend in 1 ml of PBS cntg. glucose at 0.5 - 2 x 107 cells/ml) in 15 ml conical tube, hold on ice 2. Add 5 - 50 µl (0.5 to 5 mCi) 125I using microsyringe 3. Add 50 µl of lactoperoxidase (1 mg/ml) 4. Add 10 µl of glucose oxidase (5 U/ml) 5. on ice, 5 min (occasionally agitate) 6. Add additional 10 µl of glucose oxidase (5 U/ml) 7. on ice, 5 min (occasionally agitate) 8. Add additional 10 µl of glucose oxidase (5 U/ml) 9. on ice, 5 min (occasionally agitate) 10. Add 6 ml of PBS (termination) 11. spin down the cells (1500 rpm, 3 min)* *transfer the supernatant to waste bottle containing stabilizing solution 12. Wash 2 more times with PBS 13. Suspend the cells in appropriate buffer for solubilization of cell surface proteins (successful experiments will result in 10 - 30% incorporation of 125I into cells) References 1. "Guide to radioiodination techniques"free booklet available from Amersham 2. Marchalonis, Biochem. J., 113, 299 (1969) 3. Kramer et al, Cancer Res., 49, 393 (1989) |
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