If using hard beads (i.e. PMMA, Polystyrene or Glass) follow these guidelines. This procedure can be applied to most systems that have large molecules, generally greater than 20 kDa but some smaller molecules may work. Some systems may require altered coating and blocking conditions.

1. 1. To 200 mg of hard beads (Polystyrene Part #: 442178, Glass Part # 440412, PMMA not sold by Sapidyne) add 1.0 mL of coating solution. This solution consists of 30 µg/mL of molecule in a buffer, typically 1x PBS, pH 7.4, 0.02% NaN₃. The molecule should not be reconstituted or stored in buffer containing BSA. Make sure the beads are fully suspended in solution.
   2. Rock/tumble bead vials at room temperature for 1 hour or overnight at 4°C.
   3. Allow the beads to settle or pulse centrifuge at a low speed to pellet the beads. Discard the supernatant without disturbing the settled beads.
   4. Add 1.0 mL of blocking solution. This solution consists of 10 mg/mL BSA in a buffer, typically 1x PBS, pH 7.4, 0.02% NaN₃. Make sure the beads are re-suspended in the blocking solution.
   5. Rock/tumble bead vials at room temperature for 30 min or overnight at 4°C.
   6. Beads may be used immediately or stored in the blocking solution at 4°C. If particles will not be used within one week, store at -20°C.

Notes:

• Be sure to use the “Hard Bead Handling” template for timing setup of PMMA/Polystyrene beads, and “Glass Bead Handling” for Glass beads.
• Keep all flow rates at or below 3 mL/min in the sample timing. Some customers have had success using 5 mL/min, but we do not recommend over 3 mL/min.
• Sample volumes should be kept at 5 mL or less. Some customers have had success using higher volumes, but we do not recommend volumes higher than 5 mL.
• If the molecule solution contains BSA, coating may be possible but higher concentrations could be necessary.