Fluorescein labeling of proteins

---

This method uses carboxyfluorescein succinimidyl ester (CFSE) rather than fluorescein isothiocyanate, resulting in more reliable labeling. Succinimidyl esters are excellent reagents for amine modification since the amide products formed are very stable. CFSE has high reactivity with aliphatic amines, low reactivity with aromatic amines, including tyrosine.

---

Reagents

CFSE	Carboxyfluorescein succinimidyl ester (Molecular Probes C-1311). Store desiccated at -70 °C
DMSO	Dimethylsulfoxide, anhydrous
PBS	Phosphate buffered saline, pH 7.4
	Protein to be labeled, purified, ~1 mg/ml in PBS
	Column for gel filtration, e.g. 10 ml Sephadex G-10 column
	Dialysis tubing, 10,000 MW cutoff
	Centricon microconcentrators (Amicon)

Method

1. Prepare or otherwise obtain pure protein; make sure it is free of other contaminating proteins (e.g. albumin).

2. Ensure protein to be labeled is in a suitable buffer. Buffers containing TRIS are NOT acceptable since the TRIS interferes with labeling. A reasonable buffer to use is PBS, pH 7.4. If necessary, exchange current buffer for PBS using one of three methods:
   1. Gel filtration. Do not use if you have less than 1 mg protein.
   2. Dialysis. Microdialysis is probably the best method if you do not have very much protein.
   3. Centricon microconcentrators.

3. Adjust protein concentration to ~ 1 mg/ml.

4. Prepare CFSE, 1.5 mg/ml in anhydrous DMSO. CFSE is EXTREMELY moisture sensitive! Store in desiccator at -70 °C, allow desiccator to warm to room temperature before opening. Dilute CFSE in anyhdrous DMSO immediately before use.

5. Add 100 µl CFSE per 1 ml of protein solution.

6. Incubate for 90 minutes in the dark at room temperature with continuous gentle agitation. Alternatively, incubate overnight in the dark at 4 °C with continuous gentle agitation.

7. Exact conjugation efficiency depends on temperature, length of incubation, concentration of protein, concentration of CFSE, and nature of protein. For critical applications, conjugate and test a small amount first to verify the conditions.

8. Separate labeled protein from free Fluorescein compounds by extensive dialysis versus PBS or by gel filtration. Concentrate with Centriprep and/or Centricon concentrators as necessary.

9. Microfuge on high for 10 minutes and filter through 0.22 µm filter. Optionally add 0.5% azide as preservative.

10. Determine concentration and F/P ratio by measuring absorbance at 280 nm and at 495 nm. For antibodies , use the following formulae to get approximate values:

    Protein concentration (mg/ml) = (OD₂₈₀ - 0.35 x OD₄₉₅ ) / 1.4
    F/P ratio = (3.3 x OD₄₉₅ ) / (OD₂₈₀ - 0.35 x OD₄₉₅ )