The following is a general guideline only. The optimal biotin:protein ratio must be determined by experimentation. For monoclonal
antibodies a biotin:antibody molar ratio of approximately 10 is suggested.
- Dilute or dialyze the antibody solution against 100mM NaHCO3, pH 8.3, overnight at 2 to 8° C.
- Adjust the immunoglobulin concentration to approximately 1 mg/ml.
- Dissolve the biotin ester (B0311 or B0411) in DMSO at the same concentration as the antibody.
- Add 120 µl of biotin to each 1 ml of immunoglobulin solution by slow stirring or gently vortexing (molar ratio of biotin:antibody is
approximately 40:1 to 50:1)
- Allow mixture to stir on a rotational shaker for 2 to 4 hours at room temperature.
- Dialyze against PBS (or other appropriate buffer) at 2 to 8°C to remove unreacted and hydrolyzed materials. Change buffer several times.
Alternatively, the conjugate can be buffer exchanged using a small de-salting column and analyzing the fractions for protein.
- Pass through a 0.2µm filter, determine the protein concentration, and aliquot as necessary. Conjugates may be stored refrigerated,
or frozen between -10 to -25°C, depending on the stability of the protein.
