The Thermo Scientific Pierce Magnetic IP/Co-IP Kit provides the magnetic beads, positive control and reagents to perform immunoprecipitation (IP) assays of HA-tagged proteins or co-IP reactions with HA-tagged bait proteins.
The complete Magnetic IP/Co-IP Kit can be used to perform 40 IP or Co-IP assays. It includes magnetic beads coated with immobilized anti-HA antibody, lysis/wash buffer, low pH elution buffer, neutralization buffer, HA-tag positive control lysate, and non-reducing sample buffer. Protocols are provided for both manual and automated workflows.
- Specific – highly specific anti-HA monoclonal antibody enables high yields of immunoprecipitation products
- Fast – Optimized protocol is completed in approximately 1 hour
- Low non-specific binding – stable, pre-blocked beads provide highly purified HA-tagged proteins
- Versatile – beads are compatible with manual and automated workflows (e.g., Thermo Scientific KingFisher Instruments)
- Convenient and easy – complete kit includes all necessary reagents and easy-to-follow instructions
The kit contains magnetic beads immobilized with anti-HA antibody, lysis/wash buffer, low pH elution buffer, neutralization buffer, HA-tag positive control lysate, and non-reducing sample buffer.
- High-throughput immunoprecipitation of recombinant HA-tagged proteins and co-IP of interacting proteins
The hemagglutinin (HA) peptide (YPYDVPDYA), derived from the human influenza virus HA protein, is one of several fusion protein tags used for recombinant protein expression. The Pierce Magnetic HA-Tag IP/Co-IP Kit uses a specific, high-affinity immobilized antibody for rapid purification of HA-tagged fusion proteins from bacterial and mammalian cell lysates, as well as from lysates prepared with the Pierce Human in vitro Translation Kits. The beads are incubated with a cell lysate containing HA-tagged protein, the fusion protein is captured, and the beads are subsequently washed and then eluted using low pH elution buffer or non-reducing sample buffer. The protocol and buffers have been optimized for both IP and co-IP reactions, enriching for specific protein interaction complexes in the eluted samples. Anti-HA antibody can be used to detect HA-tagged protein by Western blot analysis.
|Characteristics of Thermo Scientific Pierce Anti-HA Magnetic Beads.
||High-affinity mouse IgG1 monoclonal antibody covalently coupled to the surface of blocked magnetic beads
||Superparamagnetic (no magnetic memory)
||≥10μg of GST-ERK-HA (70kDa fusion protein)/mg of beads
||Immunoprecipitation of HA-tagged protein with the Thermo Scientific Pierce Magnetic HA-Tag IP/Co-IP Kit. HA-tagged BAD was expressed using a Pierce in vitro Expression System. Using the KingFisher Flex Instrument, 25μL each of Pierce Anti-HA Magnetic Beads or Anti-HA-tag Magnetic Beads (MBL International Corp.) were added to a 96 deep-well plate. The beads were incubated for 1 hour at room temperature with the HA-tagged BAD expressed in HeLa lysate, washed twice in IP Lysis/Wash Buffer, washed once in water and then eluted with low-pH Elution Buffer, pH 2.0. Samples were resolved by SDS-PAGE and analyzed by Western blot using the Thermo Scientific Anti-HA Monoclonal Antibody (above) and by silver stain for non-specific binding (below). The Pierce beads produced a higher yield of HA-BAD than MBL beads. Non-specific binding was minimal for both bead samples tested.
||Better co-immunoprecipitation (co-IP) with Thermo Scientific Pierce Anti-HA Magnetic Beads. HA-tagged BAD was expressed in a Pierce in vitro Expression System which includeas a HeLa lysate containing 14-3-3 proteins, a family of multifunctional regulatory proteins that bind BAD in vivo. Using a magnetic stand, 50μL each of Pierce Anti-HA Magnetic Beads and Anti-HA-tag Magnetic Beads (MBL International Corp.) were added to microcentrifuge tubes. The beads were incubated for 1 hour at room temperature with lysate containing HA-tagged BAD, washed twice with PBS containing 0.05% Tween*-20, washed once with water and then eluted in 30% acetonitrile/0.5% formic acid. Samples were dried in a Speedvac concentrator and reconstituted in reducing SDS-PAGE sample buffer. One half of each eluate was resolved by SDS-PAGE and analyzed for 14-3-3 by Western blot (above) and by silver stain (below). The Pierce Anti-HA Magnetic Beads detected co-immunoprecipitated 14-3-3 whereas the MBL beads did not. Non-specific binding was minimal for both beads tested.
Pierce Anti-HA Magnetic Beads
Other Pierce Magnetic Beads
Pierce HA-Tag IP/Co-IP Kit (agarose)
Pierce Anti-HA Agarose and Peptide
KingFisher Magnetic Particle Processors
Cell Lysis Reagents