Covalent immobilization of a molecule may result in higher signals than with adsorption coating, as well as reduce the amount of molecule necessary. If the molecule contains a free primary amine, Azlactone beads (Sapidyne Part #: 444110) can be used. The coupling is spontaneously reactive and forms a stable bond with amines (Figure 1).

1. Dry Azlactone beads should be stored at 4°C in a desiccator. Any moisture will cause the beads to hydrolize making them less efficient.
2. To a 50 mg aliquot of Azlactone beads, add 1 mL of the coating solution. This solution consists of 10-20 µg/mL molecule in 50 mM Sodium carbonate + 0.5 M Sodium citrate buffer, pH 9.0-9.6. Make sure the beads are fully suspended in solution.
3. Rock/tumble bead vials at room temperature for 1 hour or overnight at 4°C.
4. Allow the beads to settle or pulse centrifuge at a low speed to pellet the beads. Discard the supernatant without disturbing the settled beads.
5. Add 1 mL of the blocking solution. This solution consists of 10 mg/mL BSA in 1 M Tris buffer, pH 8.0-8.5. Make sure the beads are re-suspended in blocking solution.
6. Rock/tumble bead vials at room temperature for 30 minutes or overnight at 4°C.
7. Store in blocking solution at 4°C until ready for use.

Notes:

• Before starting an experiment, be sure to Edit the Bead Handling to “Soft Beads”. Keep all flow rates at or below 1 mL/min in the sample timing.
• Larger sample volumes (greater than 4 mL) should be avoided, as the flow cell screen will clog faster over time.
• Coupling is more efficient at a higher pH (9.6), provided that the protein to be coupled can withstand these conditions.
• Amine-containing buffers (Tris, Glycine, or BSA buffers) must not be used in the coupling step because their amine functional groups will compete with the protein for coupling sites.