The Thermo Scientific Pierce HA Tag IP/Co-IP Kit provides the affinity resin, positive control and other reagents necessary to perform immunoprecipitation (IP) or co-immunoprecipitation (Co-IP) reactions with an HA-tagged bait protein.
This kit is based on crosslinked beaded agarose to which a highly specific anti-HA antibody is covalently immobilized. The ready-to-use affinity resin, together with the included buffers, microcentrifuge spin columns, positive control and easy-to-follow instructions, constitute a complete set of reagents sufficient for 25 for IP or Co-IP assays. Upon incubation with a sample containing the tagged fusion protein, interaction complexes involving the HA-tagged bait protein are captured on the agarose beads. After simple washing steps, the specific protein interaction complex is easily eluted from the resin in the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis.
- Improved – updated kit includes more immobilized antibody resin and improved protocol along with GST-PI3K-SH2-HA fusion protein as a positive control
- Specific – highly specific anti-HA monoclonal antibody provides high-yield immunoprecipitation products and clean Western blot detection
- Compatible – includes protocols and reagents for multiple elution conditions to accommodate different protein sensitivities and downstream applications
- Robust – the kit and the anti-HA agarose are compatible with various cell lysates and physiologic (non-denaturing) buffer systems
- Convenient and easy – complete kit includes all necessary reagents, convenient spin columns and easy-to-follow instructions
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. Utilizing a specific, high-affinity immobilized antibody, HA-tagged fusion proteins can be quickly purified from bacterial and mammalian cell lysates as well as from the Pierce Human in vitro translation reactions. For co-immunoprecipitation reactions, simple wash steps allow enrichment and elution of specific protein interaction complexes into the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis.
||Immunoprecipitation of HA-tagged protein (GST-PI3K-SH2-HA) with the Thermo Scientific Pierce HA-Tag IP/Co-IP Kit. Coomassie Stained gel. Lane 1: MW marker, Lane 2: 15µL of 0.25mg/mL of positive control lysate, Lane 3: 15µL of Flow-through, Lane 4: 15µL of Glycine, pH 2.8 elution fraction
|Co-immunoprecipitation of active E2F1:HA-tagged pRb complex. HA-tagged pRb was expressed using a Pierce Human in vitro Expression System. 50µL of the translation reaction was diluted to 200µL in TBS and added to 15µL of agarose resin and incubated for 1 hour at 4°C as a non-specific pre-clearing step. The cleared translation reaction was added to 50µL of anti-HA resin and incubated for 3 hours at 4°C with end-over-end rocking. The resin was washed with 3 x 500µL of TBS-T. HA-pRb:E2F1 complex was eluted with the addition of 30µL of 2X non-reducing sample buffer. In vitro translation reactions without and with HA-pRb DNA (lanes 1 and 3, respectively) and anti-HA agarose elution fractions from each (lanes 2 and 4, respectively) were probed with rabbit anti-E2F1 (Part No. PA5-17549) and mouse anti-HA (Part No. 26183) and detected with Thermo Scientific SuperSignal West Dura Extended Duration Substrate (Part No. 34075).
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- Chen, Y., et al. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 6508-6512.
- Qoronfleh, M.W., et al. (2003). J. Biomed. Biotechnol. 2003(5), 291-298.
- Brymora, A., et al. (2001). Anal. Biochem. 295, 119-122.
Epitope Tag Product Selection Guide – links to antibodies, resins, magnetic beads and IP kits
Anti-HA Magnetic Beads
HA-Tag Magnetic IP/Co-IP Kit
Anti-HA Antibody and other Anti-Tag Antibodies
Fusion Protein Purification Resins and Kits
Cell Lysis and Protein Extraction Reagents