Anti-HA Magnetic Beads (1 μm)

1. Preparation 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 10 μL of Anti-HA 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 HA-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 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 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 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 4oC. 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.