The Thermo Scientific Pierce Magnetic HA-Tag IP/Co-IP Kit contains specific immunoaffinity magnetic beads and reagents to perform immunoprecipitation assays of HA fusion proteins or co-IP experiments using HA-tagged bait proteins.
Unlike traditional IP procedures based on capture with Protein A/G beads, this kit uses magnetic beads containing pre-immobilized anti-HA antibody. These Pierce Anti-HA Magnetic Beads ensure specific binding of HA-tagged protein complexes from biological samples. Because the antibody is covalently attached to the beads, IP-targets are easily eluted and recovered without antibody contamination. The complete IP kit includes the magnetic beads, lysis/wash buffer, low-pH elution buffer, neutralization buffer, HA-tag positive control lysate, and non-reducing sample buffer for SDS-PAGE. Protocols are provided for both manual and automated magnetic separation workflows. Sufficient components are provided to perform 40 IP or co-IP assays.
- Specific magnetic beads – covalently immobilized high-quality anti-HA monoclonal antibody enables high yields of immunoprecipitation products
- Low non-specific binding – stable, pre-blocked beads and specific antibody minimize off-target binding
- Trouble-free elution – low-pH elution buffer ensures recovery of HA-tagged protein interaction complexes without antibody leaching contamination
- Convenient and fast – complete kit and easy-to-follow instructions provide optimized protocols to perform IP or Co-IP experiment in approximately 1 hour
- Versatile – magnetic beads are compatible with manual and automated magnetic separation workflows (e.g., Thermo Scientific KingFisher Instruments)
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 (clone 2-2.2.14) 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.
For information about the binding capacity and other properties of the magnetic beads used in this kit, see Pierce Anti-HA Magnetic Beads (Part No. 88836).
||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.
Anti-HA Magnetic Beads
HA-Tag IP/Co-IP Kit (agarose)
Anti-HA Antibody and other Anti-Tag Antibodies
Fusion Protein Purification Resins and Kits
KingFisher Magnetic Particle Processors
Cell Lysis and Protein Extraction Reagents