Uses high-affinity antibodies to efficiently isolate c-Myc-tagged proteins and interactors.
The Thermo Scientific 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 c-Myc peptide (EQKLISEEDL) has become a popular fusion tag for mammalian recombinant protein expression. This 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
High capacity – excellent results with as little as 1µg 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
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
Product Details:
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 (5µg of immobilized c-Myc antibody) and the same amount of GST-c-Myc tag lysate sample (50µL).
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 (5µL 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 (5µg antibody in 10µL 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.
References:
Kolodziej, P.A. and Young, R.A. (1991). Methods Enzymol. 194, 508-19.
Chen, Y., et al. (1993). Proc. Natl. Acad. Sci. U.S.A. 90, 6508-12.
Qoronfleh, M.W., et al. (2003). J. Biomed. Biotechnol. 2003(5), 291-8.
Brymora, A., et al. (2001). Anal. Biochem. 295, 119-22.
