Thermo Scientific Pierce Cross-Linked Magnetic IP/Co-IP Kits use cross-linking chemistry to covalently immobilize IP antibodies in high-quality Protein A/G magnetic beads for efficient immunoprecipitation and co-immunoprecipitation.

Because this magnetic IP procedure involves the covalent binding of specific antibodies for immunoprecipitation, target proteins or co-IP protein complexes can be eluted and analyzed without the use of antibody fragments, why is that the antibody remains attached to the beads. The kit contains an optimized protocol and all the buffers and reagents necessary to obtain high-yield IP or co-IP using antibodies that bind to Protein A/G and manual or automated magnetic separation tools.

Features of Cross-linked Magnetic IP/Co-IP Kit:

•Antibody-Free IP—The antibody is irreversibly linked to the magnetic beads, making the complete antibody or containing Negligible co-elution of heavy and light chains of purified antigen
•Convenient—IP/co-IP kit contains magnetic beads and all necessary buffers for ideal immunoprecipitation
•Fast— Cross-linking and immunoprecipitation can be completed in 3 hours
•Efficient—Uses half the recommended volume of magnetic beads compared to other beads Particles for Immunoprecipitation
•Compatible— Magnetic beads coupled to Protein A/G recombinant proteins ensure compatibility with most primary antibodies, whether from mouse or rabbit
• Scalable— Use only a small amount of antibody to perform a single IP experiment or prepare larger quantities of antibody cross-linked magnetic beads for multiple experiments

Pierce Cross-Linked Magnetic IP/Co -The protocol of the IP kit is to combine the IP antibody with Protein A/G magnetic beads, and then use Disuccinimidyl Suberate (DSS) Covalently cross-links antibodies to beads. The antibody cross-linked beads are then incubated with cell lysates or tissue extracts containing the target protein antigen, allowing antigen:antibody complexes to form. The beads are washed to remove unbound material, and a low pH elution buffer is used to separate bound antigen from the antibody-cross-linked beads. Neutralizing buffer is included to prevent precipitation of isolated antigens and ensure protein activity in downstream applications. Contains lane marker sample buffer for preparing samples for SDS-PAGE analysis. This protocol is optimized for 2 to 10 µg IP antibody. For best co-IP yields, use 5 to 10 µg of antibody. Magnetic beads are removed from solution manually using a magnetic stand or through automation using an instrument such as the Thermo Scientific KingFisher Flex instrument.

The antigen yield of traditional IP (without covalent antibody linkage) is usually higher than that of IP schemes using cross-linking. This is because the cross-linking process inevitably causes the loss of some functional antibody binding sites. To overcome this limitation of the cross-linking method, the amount of IP antibody required in the cross-linking IP method may be greater than in an equivalent traditional IP procedure. Of course, the advantage of the cross-linked immunoprecipitation procedure is that there are no interfering antibody bands on the Western blot.

Protocol Summary
1: Pre-wash the beads with 1X coupling buffer.
2: Let the antibody bind to the protein A/G magnetic beads for 15 minutes.
3: Wash the beads three times with 1X coupling buffer.
4: Use DSS to cross-link the antibody to the beads for 30 minutes.
5: Wash the beads three times with elution buffer, then twice with IP lysis/wash buffer.
6: Incubate the cell lysate with the prepared beads at room temperature for 1-2 hours or overnight at 4°C.
7: Wash the beads twice with IP lysis/wash buffer and then once with purified water.
8: Elute bound antigen.

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