Perform IP or Co-IP experiments with a c-Myc tagged protein.

The c-Myc peptide (EQKLISEEDL) has become a popular fusion tag for mammalian recombinant protein expression. The Pierce c-Myc Tag IP/Co-IP Kit provides the affinity resin and other reagents necessary to easily perform immunoprecipitation (IP) or co-immunoprecipitation (co-IP) experiments using a c-Myc-tagged protein as the "bait". The kit includes crosslinked beaded agarose to which a highly specific anti-c-Myc antibody is covalently immobilized. Upon incubation with a sample containing the tagged fusion protein, interaction complexes involving the c-Myc-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-c-Myc monoclonal antibody binds the c-Myc 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 1 µg of anti-c-Myc antibody in IP mode with the positive control lysate
• Rigorous – kit includes a positive control lysate that contains over-expressed GST-c-Myc 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 physiological (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-c-Myc antibody preparations. The antibody agarose resin in the Pierce IP/Co-IP Kit (Pierce) outperforms other commercially available standalone preparations (Vendors A-D). Each IP was conducted with the same amount of antibody (5 µg of immobilized c-Myc antibody) and the same amount of GST-c-Myc tag lysate sample (50 µl).

Figure 2. Co-IP of SV40 large T-antigen (LTA) with c-Myc-tagged p53. LTA and c-Myc-p53 were expressed and 35S-labeled in vitro (Lane 1 and 2). Before IP and co-IP, the lysates (5 µl each) of LTA and c-Myc-p53 (Lanes 3 and 6), c-Myc-p53 alone (Lane 4) or LTA alone (Lane 5) were incubated at 30°C for 1 hour. Then anti-c-Myc agarose (5 µg antibody in 10 µl slurry) (Lanes 3, 4, 5) or plain agarose slurry (Lane 6) was added to the corresponding sample and incubated overnight at 4°C. Finally, IP and co-IP products were eluted, separated by 12% SDS-PAGE and the 35S-labeled proteins detected by fluorography.

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.