Protocol
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).
1.2 Transfer 25-50 μL of Protein A/G Magnetic Beads into a 1.5 mL tube (Transfer amount may be adjusted as required).
1.3 Add 400 μL of binding/wash buffer to the beads and gently pipette to mix. Place the tube into a magnetic stand to collect the beads against the side of the tube (Hereinafter referred to as magnetic separation). Remove and discard the supernatant. Repeat this step for 2 times.
Binding of Antibody
2.1 Dilute antibody (Ab) to the final concentration of 5-50 μg/mL with binding/wash buffer. The optimal amount of Ab may be adjusted as required.
2.2 Add 400 μL of diluted Ab to the Protein A/G Magnetic Beads. Rotate tube for 30 minutes at room temperature or 2 hours at 4°C.
2.3 Perform magnetic separation. Transfer the supernatant into a new tube for further analysis, if desired. The supernatant is the non-binding fraction.
2.4 Add 400 μL of binding/wash buffer to the beads and gently pipette to mix. 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.
Immunoprecipitation of Target Antigen
3.1 Remove the tubes from the magnetic separator and add your sample containing the antigen (Ag) (typically 5-50 μg in 400 μL binding/wash buffer) and gently pipette to resuspend the Protein A/G Magnetic Beads-Ab complex.
3.2 Incubate with rotation for 30 minutes at room temperature or 2 hours at 4°C to allow Ag to bind to the Protein A/G Magnetic Beads-Ab complex.
3.3 Perform magnetic separation. Remove and discard the supernatant.
3.4 Wash the Magbeads-Ab-Ag complex 5 times using 400 μL binding/wash buffer for each wash. Perform magnetic separation between each wash, remove supernatant and resuspend by gentle pipetting.
3.5 Resuspend the Protein A/G Magnetic Beads-Ab-Ag complex in 400 μL binding/wash buffer and transfer the bead suspension into a clean tube. This is recommended to avoid co-elution of the proteins bound to the tube wall.
Elution
This is a non-denaturation elution method.
4.1 Perform magnetic separation and remove the supernatant. Add 400 μL of binding/wash buffer into the tube and rotate for 5 minutes. Perform magnetic separation for 1 minute and remove the supernatant. Then add 25-50 μL elution buffer into the tube with magnetic beads-Ab-Ag complex, rotate for 5 minutes.
4.2 Perform magnetic separation, collect the supernatant.
4.3 The final solution can be used as samples for denaturing SDS-PAGE. Or the elution can be adjusted to neutral pH with neutralization buffer immediately and used for further analysis.
