Anti-Flag Magnetic Beads

1. reparation of Magnetic Beads

1.1 Resuspend the Magnetic Beads in the vial (tilt and rotate for 2 minutes or gently pipette for 10 times, do not vortex). Transfer 20 μL of Anti-Flag Magnetic Beads suspension into a new tube.

1.2 Add 500 μL of wash buffer to the beads and gently pipette to mix. Place the tube into a magnetic stand (MCE Cat. No.: HY-K0200) to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step for 2 times

2. Protein Binding

2.1 Add 500 μL of cell lysate (the sample containing Flag-tagged protein) to the washed beads. For Ag binding, incubate for 2 hours at room temperature or overnight at 4°C while gently rotating the tube.

2.2 Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant.

NOTE: Occasional aggregation of magnetic beads during the binding process doesn’t affect experimental results.

3. Washing

Add 500 μL of wash buffer to the Magbeads-Ag complex and mix gently. Place the tube into a magnetic stand to collect the beads against the side of the tube. Remove and discard the supernatant. Repeat this step for 4 times.

4. Elution & Detection

Three elution methods are recommended according to protein characteristics or further usage:

1) Elution with sample buffer for gel electrophoresis and immuoblotting. Add 50 μL of 1× SDS-PAGE loading buffer to each tube and boil for 5 minutes. Cool and place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. Keep the supernatant containing the target antigen for SDS-PAGE analysis

2) Elution with Elution Buffer A (0.15 M Glycine, pH 2.5-3.1) under acidic condition. Add 50 μL of Elution Buffer A to each tube. Incubate with gentle shaking or on a rotator for 10 minutes at room temperature. Place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. Adding 25 μL of Neutralization Buffer (1 M Tris-HCl, pH 8.0) for each 50 μL of eluate to neutralize the low pH, which may help preserve bioactivity of target protein.

3) Elution with Elution Buffer B (1 mg/mL 3× Flag peptide, 50 mM Tris, 0.15 M NaCl, pH 7.4) under native condition. Add 3-5 (v/v) volume of Elution Buffer B to each tube. Incubate with gentle shaking or on a rotator for 1 hour at room temperature or 2 hours at 4°C. Place the tube into a magnetic stand to collect the beads and transfer the supernatant to a new tube. For immediate use, store the eluates at 4°C, or store at -20°C for long term storage.