Perform IP or Co-IP experiments with an HA-tagged protein.

The hemagglutinin (HA) peptide (YPYDVPDYA) has become popular fusion tag for mammalian recombinant protein expression. Pierce HA Tag IP/Co-IP Kit provides the affinity resin and other reagents necessary to easily perform immunoprecipitation (IP) or co-immunoprecipitation (co-IP) reactions using an HA-tagged protein as the "bait". The kit includes crosslinked beaded agarose to which a highly specific anti-HA antibody is covalently immobilized. 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. The kit includes the prepared agarose affinity resin, buffers, microcentrifuge spin columns, a positive control and easy-to-follow instructions.

Highlights:

• Specific – the immobilized anti-HA monoclonal antibody binds the HA epitope tag with high specificity, providing high yield immunoprecipitation products and clean Western blot detection (Figures 1 and 2)
• High Capacity – excellent results with as little as 2.5 µg of anti-HA antibody in IP mode with the positive control lysate
• Rigorous – kit includes a positive control lysate that contains over-expressed GST-HA to validate the reagents and specific protocol, and the instructions provide tips for planning other controls (see Figure 2 for example controls)
• Robust – the affinity system is compatible with IP or Co-IP from various cell lysates and physiologic (non-denaturing) buffer systems; choose the Mammalian Kit to use the validated M-PER Mammalian Cell Lysis Reagent for the procedure
• Convenient and Easy – complete kit includes all necessary reagents, convenient spin columns, and easy-to-follow instructions

Figure 1. Immunoprecipitation results with different immobilized anti-HA antibody preparations. The antibody agarose resin in the Pierce IP/Co-IP Kit (Pierce) outperforms other commercially available preparations (Vendors A-C). Each IP was conducted with the same amount of antibody (10 µg of immobilized HA antibody) and the same amount of GST-HA tag lysate sample (50 µl).

Panel A		Panel B
Figure 2. Co-IP of active Rac1 with HA-tagged Pak1-PBD (p21 binding domain). Panels are the same Western blot probed first with anti-Rac1 antibody (Panel A), then stripped and probed with anti-HA antibody (Panel B). Human 293 cells were transfected with HA-Pak1-PBD alone or co-transfected with constitutively activated Rac1 (Q61L). Anti-HA agarose slurry (6 µl) was incubated with 50 µl of HA-tagged positive control lysate from the kit (Lane 1) or 500 µl of the co-transfected cell lysate (Lane 2). HA-Pak1-PBD transfected cells (Lane 3) and non-transfected cells (Lane 4) represent two kinds of control conditions that are useful in such experiments. IP and co-IP binding reaction were performed overnight at 4°C.

Related Products:
Other Co-IP and Pull-Down Kits
Immunoprecipitation (IP) Kits

References:

1. Kolodziej, P.A. and Young, R.A. (1991). Methods Enzymol. 194, 508-519.
2. Chen, Y., et al. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 6508-6512.
3. Qoronfleh, M.W., et al. (2003). J. Biomed. Biotechnol. 2003(5), 291-298.
4. Brymora, A., et al. (2001). Anal. Biochem. 295, 119-122.