The Thermo Scientific Pierce Crosslink Magnetic IP/Co-IP Kit uses crosslinking chemistry to covalently immobilize IP antibodies onto premium-quality Protein A/G Magnetic Beads for effective immunoprecipitation and co-immunoprecipitation.
Because this magnetic IP procedure involves covalent attachment of the specific antibody used for immunoprecipitation, target proteins or co-IP protein complexes can be eluted and analyzed without antibody fragments because the antibody remains affixed to the beads. The kit contains an optimized protocol and all buffers and reagents necessary to accomplish high-yield IP or co-IP with antibodies that bind to Protein A/G and using either manual or automated magnetic separation tools.
- Antibody-free IP – antibody is irreversibly attached to the magnetic beads resulting in negligible co-elution of intact antibody or heavy and light chains with the purified antigen
- Convenient – the IP/co-IP kit contains magnetic beads and all essential buffers for optimal immunoprecipitation
- Fast – complete crosslinking and immunoprecipitation in less than 3 hours
- Efficient – immunoprecipitate with half the recommended volume of magnetic particles compared to other magnetic beads
- Compatible – magnetic beads coupled with Protein A/G recombinant protein ensures compatibility with most primary antibodies, whether from mouse or rabbit
- Scalable – use only the amount of antibody needed for a single IP experiment or prepare a larger quantity of antibody-crosslinked magnetic beads for multiple experiments
Pierce Protein A/G Magnetic Beads, DSS crosslinker and coupling buffer, lysis/wash buffer, elution buffer, neutralization buffer and sample loading buffer
The protocol for the Pierce Crosslink Magnetic IP/Co-IP Kit binds IP antibody to Protein A/G Magnetic Beads and then covalently crosslinks the antibody to the beads using disuccinmidyl suberate (DSS). The antibody-crosslinked beads are then incubated with cell lysate or tissue extract that contains the protein antigen of interest, allowing the antigen:antibody complex to form. The beads are washed to remove non-bound material and a low pH elution buffer is used to dissociate bound antigen from the antibody-crosslinked beads. Neutralization buffer is included to prevent precipitation of the isolated antigen and to ensure protein activity in downstream applications. Lane Marker Sample Buffer is included for preparing samples for SDS-PAGE analysis. The protocol is optimized for 2 to 10µg of IP antibody. For optimal co-IP yields, use 5 to 10µg of antibody. 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.
Traditional IP (without covalent antibody attachment) generally provides higher antigen yields than IP protocols that used crosslinking. This is because the crosslinking process inevitably causes loss of some functional antibody binding sites. To overcome this limitation with the crosslink method, it may be necessary to use somewhat greater amounts of IP antibody in the crosslink IP method than in the equivalent traditional IP procedure. Of course, the advantage with the crosslink immunoprecipitation procedure is Western blots devoid of interfering antibody bands.
- Prewash beads with 1X Coupling Buffer.
- Bind antibody to Protein A/G magnetic beads for 15 minutes.
- Wash beads three times with 1X Coupling Buffer.
- Crosslink antibody to beads with DSS for 30 minutes.
- Wash bead three times with Elution Buffer and then twice with IP Lysis/Wash Buffer.
- Incubate cell lysate with prepared beads for 1-2 hours at room temperature or overnight at 4°C.
- Wash beads twice with IP Lysis/Wash Buffer and then once with purified water.
- Elute bound antigen.
|The Thermo Scientific Pierce Crosslink Magnetic IP /Co-IP Kit immunoprecipitates PP2A without antibody contamination and with negligible background. PP2A antibody (5µg) was coupled to Pierce Protein A/G Magnetic Beads without DSS crosslinking (traditional IP) and with DSS crosslinking (Crosslink IP). The beads were incubated with 0.5mg of A549 cell lysate for 1 hour at room temperature on the Thermo Scientific KingFisher Flex Instrument. The beads were washed twice with IP Lysis/Wash Buffer and once with water. PP2A was eluted from the beads with Elution Buffer for 5 minutes at room temperature and then neutralized with Neutralization Buffer. The eluates, antibody control (Ab) and flow-through (FT) were resolved by SDS-PAGE and analyzed by Western blot for PP2A (Panel A) and by silver stain for antibody contamination and non-specific binding (Panel B). The antibody-crosslinked magnetic beads effectively immunoprecipitated PP2A without antibody contamination (the same antibody was used for both IP and Western blot detection to detect the presence of antibody) whereas the traditional IP method resulted in significant antibody contamination in the eluate.
Review of Immunoprecipitation
Immunoprecipitation kit selection guide
Pierce Classic Magnetic IP/Co-IP Kit – Protein A/G kit without the crosslinking function
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