Coupling Protocol B: Two-Step Coupling Supplemented with Sulfo-NHS
(Similar to Protocol A but uses Sulfo-NHS to stabilize intermediates and enhance efficiency)

1. Ensure protein/ligand is in amine-free coupling buffer. Use 50–400 μg protein per 100 μL coupling buffer. Keep on ice.

2. Vortex carboxyl beads to resuspend. Transfer 100 μL beads to a 1.5 mL tube. Separate magnetically and remove supernatant.

3. Add 200 μL coupling buffer, vortex vigorously for 20 sec. Separate magnetically and remove supernatant.

4. Repeat wash (Step 3) twice more.

5. Prepare fresh solutions in coupling buffer: EDC (50 mg/mL) and Sulfo-NHS (50 mg/mL).

6. Add 60 μL coupling buffer, 20 μL fresh EDC solution, and 20 μL fresh Sulfo-NHS solution to the beads.

7. Mix and incubate at RT for 15 min. Separate magnetically and remove supernatant.

8. Wash PuriMag™ beads with 200 μL coupling buffer. Vortex to mix. Separate magnetically and remove supernatant.

9. Add 100 μL coupling buffer and 50–400 μg protein/ligand. Vortex to mix. Incubate at RT for 0.5–4 h.

10. Place tube in magnetic stand, separate magnetically, and remove supernatant (Save for analysis if optimizing).

11. Add 250 μL quenching buffer to beads. Vortex for 20 sec. Separate magnetically and remove supernatant.

12. Add 500 μL quenching buffer to beads. Incubate at RT for 30–60 min. Separate magnetically and remove supernatant.

13. Add 250 μL quenching buffer to beads. Vortex vigorously for 20 sec. Separate magnetically and remove supernatant.

14. Remove tube from magnetic stand. Add 100 μL storage buffer. Vortex to mix and store beads at 2–8°C.