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.
|Ab||Antibody, about 2.5 mg/ml|
|CFSE||Carboxyfluorescein succinimidyl ester (Molecular Probes C-1311). Store desiccated|
|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)|
- Prepare or otherwise obtain pure protein; make sure it is free of other contaminating proteins (e.g. albumin).
- 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:
- Gel filtration. Do not use if you have less than 1 mg protein.
- Dialysis. Microdialysis is probably the best method if you do not have very much protein.
- Centricon microconcentrators.