Which bifunctional ligand should I use?

The choice of bifunctional ligand depends on the type of functional group that you plan to use for covalent attachment. We currently offer isothiocyanate and NHS-esters for attachment to primary amines, maleimides for attachment to thiols, and amine functionalized bifunctionals that can be attached to electrophilic groups. For conjugation reactions performed under aqueous conditions B-205, B-305, and B-280 are representative examples of some very popular bifunctional ligands based on isothiocyanates and NHS-ester linkages :

We also carry a variety of other bifunctional ligands that are specifically designed for optimal performance with different metals and for use in solid phase and organic solvent applications.

What are typical conditions for preparing lanthanide complexes?

Typically, the bifunctional ligand is first conjugated to a protein, peptide, or an antibody prior to complexation. The key to successful complexation is to make sure the conjugate is adequately buffered at a slightly acidic pH prior to introduction of the lanthanide. If the metal experiences a basic pH upon addition, it is likely to form the hydroxide species [Ln(OH)₃] which undergoes complexation at a much slower rate relative to the LnCl₃ species. Formation of the hydroxide can be detected visually by the appearance of a slightly turbid suspension. Ultimately this condition leads to a reduce percentage of complexation.

What can I do to increase conjugation efficiency?

Isothiocyanate bifunctional ligands react quickly with primary amines at neutral to moderately basic conditions whereas the NHS-ester begin to reacts under slightly acidic conditions. Both will undergo competitive hydrolysis under these same conditions which reduces conjugation efficiency. For this reason it is important to combine the bifunctional ligand with the targeting vector in a properly buffered medium and to avoid preparing the bifunctional ligand solution too far in advance. It is also advantageous to use an excess of bifunctional ligand as some degree of hydrolysis is unavoidable.