Direct labelling of antibodies with fluorescent dyes
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| Materials | |
|---|---|
| Pacific Blue succinimidyl ester (absMAX= 416nm; emMAX= 451nm) | |
| 5-(6)-carboxyfluorescein succinimidyl ester (FAM, SE) (absMAX= 494nm; emMAX= 518nm) | |
| 5-(6)-carboxytetramethylrhodamine succinimidyl ester (TAMRA, SE) (absMAX= 555nm; emMAX= 580nm) | |
| AlexaFluor-647 succinimidyl ester (absMAX= 650nm; emMAX= 665nm) 1 x phosphate-buffered saline (PBS) | |
| 1M sodium bicarbonate (pH 8.5) | |
| STOP buffer (prepare fresh): 1.5 M hydroxylamine hydrochloride, pH 8.5 (dissolve 630 mg in 5 ml distilled H20; adjust pH to 8.5 with 1M NaOH, fill up with H20 to 6 ml) | |
| Float-A-Lyzer G2 dialysis chamber (SpectraPor), 100 kDa, 1 ml volume | |
| 1 x phosphate-buffered saline (PBS) |
Procedure
- Take purified monoclonal antibody (e.g. anti-CEACAM, clone 18/20; control IgG1, clone 96/1) and dilute 500 µg antibody in a total of 500 µl PBS.
- Add 50 µl of 1M sodium bicarbonate (pH 8.5) to 500 µl antibody in PBS. Incubate on Mixer at RT.
- Add 50 µl of Pacific Blue (or other dye) (stock aliquots are at 2 mg/ml in DMSO)
- Incubate for 60 min at RT with shaking (protected from light)
- Add 100 µl of STOP buffer, incubate 1 hour at RT with shaking (protected from light)
- Add antibody-dye solution to Float-A-Lyzer G2 dialysis chamber and dialyse o/n at 4°C in 1 liter PBS (keep dark w/ aluminium foil), change PBS twice during the next morning (incubate for a total of 4 h at 4°C).
- Check labeling and protein concentration with spectrofluorometer (Pacific Blue: absMAX= 416nm; emMAX= 451nm)
- Add to the dialyzed, dye-coupled antibody:
- azide (0,05% final conc.), BSA (0.1 mg/ml final conc.), and glycerol (20% final conc.)
- Aliquot in 25 µl aliquots and freeze at -80°C