The Thermo Scientific Pierce Classic Magnetic IP/Co-IP Kit enables highly effective and efficient immunoprecipitation (IP) and co-immunoprecipitation (co-IP) of antigens and protein-complexes using less than 10µg of antibody and a magnetic separator.
This immunoprecipitation kit uses high-quality Thermo Scientific Pierce Protein A/G Magnetic Beads and optimized buffers and flexible protocol to accomplish high-yield IP or co-IP with manual or automated magnetic-separation tools. The optimized Lysis/Wash Buffer optimizes yield and efficient binding of antibody-antigen and co-IP interactions. The relatively gentle, low-pH Elution Buffer dissociates the bound immune complex from the Protein A/G. Alternatively, the included Lane Marker Sample Buffer provides rapid, denaturing elution for direct SDS-PAGE analysis of IP products.
- Compatible – magnetic beads based on Protein A/G recombinant protein ensures compatibility with most primary antibodies, whether from mouse or rabbit
- Fast – immunoprecipitate in as few as 30 minutes to reduce background and improve the capture of transient protein complexes
- Clean – immobilize your antibody to prevent contamination in your eluate
- Resistant – no leaching of Protein A/G in the presence of detergents, low pH buffers or common mass spectrometry solvents
- Efficient – immunoprecipitate with half the recommended volume of magnetic particles compared to other magnetic beads
- Convenient – the comprehensive Co-IP kit contains the magnetic beads and all essential buffers to complete immunoprecipitation experiments
Pierce Protein A/G Magnetic Beads, lysis/wash buffer, elution buffer, neutralization buffer and sample loading buffer
Immunoprecipitation with magnetic particles is performed much like IP with beaded agarose, except that separations are performed using a magnet rather than by centrifugation. The specific antibody is first added to the sample to form an immune complex that is then bound to the magnetic beads. The complex is washed to remove non-bound material and a low-pH elution buffer dissociates the bound immune complex from the Protein A/G. Alternatively, the Lane Marker Sample Buffer is included for dissociation using denaturing conditions or for downstream sample prep for SDS-PAGE analysis. The kit includes Thermo Scientific Pierce Protein A/G Magnetic Beads for fast and convenient magnetic isolation of antigens and optimized buffers for high antigen yield. The beads are removed from the solution manually using a magnetic stand or by automation with an instrument such as the Thermo Scientific KingFisher Flex Instrument.
It is generally known that longer antibody-sample incubations times (2 hours to overnight) usually provide greater yields in IP assays. It is often assumed that this greater yield comes with increased background. Our researchers have found that overnight antibody-sample binding followed by immunoprecipitation with the Pierce Classic Magnetic IP/Co-IP Kit does tend to increase yield but does not increase background. Thus, we recommend that researchers perform this binding step for at least 1 hour whenever possible.
- Incubate cell lysate with IP antibody for 1 to 2 hours at room temperature or overnight at 4ºC.
- Bind antigen-antibody complex to Protein A/G magnetic beads for 1 hour at room temperature.
- Wash beads twice with IP Lysis/Wash Buffer and once with purified water.
- Elute the antigen/antibody complex.
|Higher IP yield with Protein A/G beads compared to Protein A or Protein G beads. Thermo Scientific Pierce Protein A/G Magnetic Beads (Classic Magnetic IP Kit) immunoprecipitate Cdk1 with greater yield than other brands of separate Protein A and Protein G magnetic beads.
U2OS (human osteosarcoma) cells were synchronized in late G2 phase of the cell cycle by serum starvation followed by growth in 20% fetal bovine serum for 18 hours prior to harvest. The cells were lysed in IP Lysis/Wash Buffer, and 0.75mg of lysate (per sample) was incubated with and without anti-Cdk1 antibody overnight at 4°C. Pierce Protein A/G Magnetic Beads were compared to Mag Sepharose™ Beads (GE Healthcare), Dynabeads™ (Life Technologies) and PureProteome™ (Millipore) Protein A and Protein G products. Respective brands of magnetic beads were added (50µL each) to a 96 deep well plate. Using the Thermo Scientific KingFisher Flex Instrument, the beads were washed with IP Lysis/Wash Buffer incubated for 1 hour with antigen sample/antibody mixture, washed twice with IP Lysis/Wash Buffer containing 0.5M NaCl, washed once with water, and then eluted with SDS-PAGE reducing sample buffer for 10 minutes at room temperature. The eluates were resolved by SDS-PAGE and analyzed by Western blot for Cdk1.
|Co-immunoprecipitation of cyclin B and Cdk1. The Thermo Scientific Pierce Protein A/G magnetic beads bind to Cdk1 antibody complexed with Cdk1. Cyclin B is bound to the Cdk1, and is captured along with its binding partner.
|The Thermo Scientific Pierce Classic Magnetic IP Kit co-immunoprecipitates (co-IP) cyclin B and Cdk1 with negligible background. U2OS cells were synchronized and samples were prepared as described in the figure above. The eluates were resolved by SDS-PAGE and either analyzed by Western blot (Panel A) for cyclin B or silver stained (Panel B). The Pierce Protein A/G beads were found to effectively co-IP cyclin B with a higher yield than Mag Sepharose™ Beads (GE Healthcare) and PureProteome™ Beads (Millipore), and equivalent yield to Dynabeads™ Beads (Life Technologies) (Panel A). The Pierce beads bound the same or more antibody than the other brands of Protein A and Protein G magnetic beads (Panel B). All beads were found to have negligible non-specific binding.
Review of Immunoprecipitation
Immunoprecipitation kit selection guide
Pierce Crosslink Magnetic IP/Co-IP Kit – covalently attaches IP antibodies to Protein A/G beads
Pierce Direct Magnetic IP/Co-IP Kit – directly immobilizes IP antibodies without using Protein A/G
Pierce Magnetic Beads – Protein A/G, NHS ester, Streptavidin, etc.
Thermo Scientific KingFisher Magnetic Particle Processors