• FITC Labeling of Proteins1)
  • The following protocol can be used to label proteins with concentrations 2-10mg/ml

1. Set the pH of the protein solution to 9.3 via dialysis against 2L of 100mM Sodium Carbonate at 4^oC for 24 hrs
   • Change the dialysis solution once during this process
2. Measure OD₂₈₀ of protein solution
   • Calculate Molar Concentration ([Protein] M) using OD₂₈₀/extinction coefficient of Protein
3. Weigh out 3-4 mg of FITC and add it to 1 mL of 100 mM Sodium Carbonate pH 9.3
4. Dilute FITC solution 100x with 100mM Sodium Carbonate pH 9.3
   • Use stock and diluted FITC solution within 1-2 hrs
5. Measure OD₄₉₅ and OD₂₈₀ of dilute FITC solution. Record rFITC = OD₄₉₅/OD₂₈₀
   • rFITC should be between 3 and 3.3
6. Calculate Molar Concentration of FITC ([FITC] M) = OD₄₉₅/extinction coefficient at pH 9.3 (73,000)
7. Add 15-20ug of FITC per mg of protein. If [protein] is less than 5mg/ml, [FITC] should not be lower than 100ug/ml
8. Incubate for 1 hr at room temperature or overnight at 4^oC
9. To purify, use a Sephadex G25 Column primed with 1x PBS pH 7.4
10. The first eluent with a greenish tint should be collected and saved as this is the labeled protein
11. Calculate labeling ratio as follows:
    • [Protein] mg/ml = (OD₂₈₀ FITC-Protein - ((OD ₄₉₅ FITC-Protein)/rFITC)/1.4
    • [Protein] M = ([Protein] mg/ml)/MW Protein
    • [FITC] M = (OD₄₉₅ FITC-Protein)/FITC coefficient of extinction at pH 7.4 (63,000)
    • Labeling Ratio F/P = ([FITC] M)/([Protein] M)
12. For Best Results F/P should be between 2 and 4, 1 and 5 are acceptable, but not ideal

Edited by Mitchell Weiser 3/1/07