Anti-Flag Magnetic Agarose Beads

Recommended Buffers (Not Provided)

Note: Prepare all buffers with ultrapure water and sterilize them by filtration through a 0.45 μm or 0.22 μm membrane.

Protein Purification/Immunoprecipitation

1. Bead Pretreatment: Thoroughly resuspend the Anti-Flag Magnetic Agarose Beads. Transfer the required volume into a 1.5 mL tube, add 500 μL of binding/wash buffer, mix thoroughly, perform magnetic separation, and discard the supernatant. Repeat 2-3 times.

2. Sample Binding: Add 200-1,000 μL of clarified sample and incubate on a rotator at 4°C for 2 h or overnight. Perform magnetic separation and transfer the supernatant to a new tube for analysis if required.

3. Washing: Add 500 μL-1 mL of binding/wash buffer, resuspend thoroughly, perform magnetic separation, and discard the supernatant. Repeat at least three times, or until the OD₂₈₀ of the wash supernatant is below 0.05.

4. Elution: Select acidic, competitive, or denaturing elution. For acidic elution, add 50-100 μL of acidic elution buffer, incubate at room temperature for 5-10 min, and neutralize immediately. For competitive elution, add 50-100 μL of competitive elution buffer and incubate at room temperature for 10-30 min. For denaturing elution, add 20-50 μL of 2× SDS-PAGE Loading Buffer, heat at 95°C for 5 min, perform magnetic separation, and collect the supernatant.

Note: Acid-eluted proteins may be stored at 4°C for a short period or at -20°C for long-term storage. After denaturing elution with SDS, β-mercaptoethanol, or DTT, the immobilized antibody or ligand may be inactivated and the beads should not be reused.

5. Storage: If the beads will be reused, wash thoroughly with binding/wash buffer, resuspend in storage buffer, and store at 2-8°C. Do not freeze, dry, or expose the beads to a magnetic field for an extended period.