Bioradiations Staff
Introduction
Follow this simple protocol for immunoprecipitation using Bio-Rad’s SureBeads Magnetic Beads. SureBeads Magnetic Beads contain a superparamagnetic core, which enables rapid and highly efficient immunoprecipitation without the need for centrifugation. SureBeads Protein G Magnetic Beads and Protein A Magnetic Beads, and the SureBeads Magnetic Racks are designed for quick and easy immunoprecipitation (IP) and protein complex pull-down.
Preparation and Binding
- Thoroughly resuspend the SureBeads in their solution and transfer 100 µl (1 mg at 10 mg/ml) of SureBeads to 1.5 ml tubes. Magnetize beads and discard supernatant.
- Wash with 1,000 µl PBS-T (PBS + 0.1% Tween 20):
- Resuspend the beads thoroughly
- Magnetize beads and discard supernatant, repeat three times (3x).
Tip: Resuspension can be achieved by vortexing or pipetting while the magnet slider is outside of the tube holder. If vortexing, spin down the tubes before magnetization to bring down drops from the tube cap.
- Add 1–10 µg antibody in final volume of 200 µl and resuspend the beads.
- Rotate 10 min at room temperature (RT).
- Magnetize beads and discard supernatant.
- Wash with 1,000 µl PBS-T:
- Resuspend the beads thoroughly
- Magnetize beads and discard supernatant, repeat three times (3x).
- Add the antigen-containing lysate, 100–500 µl.
- Rotate for 1 hr at room temperature (RT).
- Magnetize beads and discard supernatant.
- Wash with 1,000 µl PBS-T:
- Resuspend the beads thoroughly
- Magnetize beads and discard supernatant, repeat three times (3x). Before the last magnetization, transfer the resuspended beads to a new tube.
- Spin down all tubes for several seconds.
- Magnetize beads and aspirate residual buffer from the tubes.
Elution
Elution Strategy 1:
- Add 20 µl glycine 20 mM pH 2.0 and incubate 5 min at RT
- Magnetize beads and move eluent to a new vial
- Neutralize eluent with 2 µl (10% eluent volume) 1 M phosphate buffer pH 7.4.
Elution Strategy 2:
- Add 40 µl 1x Laemmli buffer and incubate for 10 min at 70°C
- Magnetize beads and move eluent to a new vial.
Ordering Information
Catalog # |
Description |
| 1614013 | SureBeads Protein A Magnetic Beads, 3 ml |
| 1614023 | SureBeads Protein G Magnetic Beads, 3 ml |
| 1614916 | SureBeads Magnetic Rack, 16 tube holder |
| 1614833 | SureBeads Starter Kit AG, 3 ml protein A beads, 3 ml G beads and a 16 tube magnetic rack |
| 1614813 | SureBeads Starter Kit AA, two 3 ml protein A beads and a 16 tube magnetic rack |
| 1614823 | SureBeads Starter Kit GG, two 3 ml protein G beads and a 16 tube magnetic rack |
| 1614811 | SureBeads Trial Kit Protein A |
| 1614821 | SureBeads Trial Kit Protein G |
Visit www.bio-rad.com/SureBeadsIP for more information on SureBeads magnetic beads.
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