Radioiodination of cell surface proteins

Friday, October 17, 2003

Description
Radioiodination of cell surface proteins

Procedure
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 l (0.5 to 5 mCi) 125I using microsyringe
3. Add 50 l of lactoperoxidase (1 mg/ml)
4. Add 10 l of glucose oxidase (5 U/ml)
5. on ice, 5 min (occasionally agitate)
6. Add additional 10 l of glucose oxidase (5 U/ml)
7. on ice, 5 min (occasionally agitate)
8. Add additional 10 l 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)

Recipes
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

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Radioiodination of cell surface proteins