Fluorescent Conjugation of Biomolecules

Cy3-conjugated ovalbumin (OVA) (Sigma) and Cy3-conjugated chicken gamma globulin (Rockland) were prepared as previously described (29 (link)). 5-HIAA (Sigma) or IPA (Sigma) conjugated allophycocyanine (APC) (Prozyme), phycoerythrin (PE) (Prozyme), or BSA (Sigma) were generated using the Mannich condensation reaction. Briefly, 10 μM APC, PE, or BSA was mixed with 2.7 mM 5-HIAA or IPA in 0.25 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.5) with 500 mM NaCl, 2 mM vitamin C (Sigma), and 3.5% final concentration of formaldehyde (plus 10% methanol to prevent polymerization) for 24 h at 37 °C. All procedures were protected from light. Unbound 5-HIAA or IPA was removed using a 10 KD cutoff spin column (Millipore). Control APC, PE, or BSA was made using the same conjugation protocol but without the addition of 5-HIAA or IPA.