1. 5(6)-Carboxyfluorescein-N-hydroxysuccinimide ester (Roche Diagnostics, Lewes, UK; Molecular Probes, Eugene, OR) and 5(6)-carboxytramethylrhodamine-N-hydroxysuccinimide ester (Molecular Probes, Eugene, OR) are alternative reagents. They can be conjugated to IgG in PBS at room temperature in about 1 h.
2. Ready-made Sephadex G-25 columns are available for use with gravity feed (PD-10) or syringe (HiTrap) from Amersham Biosciences. The PD-10 columns with a bed volume of approx 9 mL are very suitable for processing samples of 1-2 mL. Similar cross-linked dex-tran devices, Presto and Kwik columns, are available from Pierce (Rockford, IL).
3. The BSA minimizes losses due to adsorption and denaturation.
4. The extent of the conjugation can be determined by absorbance measurements. The absorbance maxima of fluorescein or rhodamine (495 and 555 nm, respectively) are related to the IgG absorbance at 280
nm. Ratios of 0.4-1.0 are appropriate for fluorescein conjugates and 0.3-0.9 for rhodamine conjugates. (These measurements must be made before the addition of BSA.)
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