Perform co-immunoprecipitation experiments without antibody interference.

The Thermo Scientific Pierce Co-Immunoprecipitation (Co-IP) Kit enables isolation of native protein complexes from a lysate or other complex mixture by directly immobilizing purified antibodies onto an agarose support.

Co-IP is a common approach to study protein:protein interactions that uses an antibody to immunoprecipitate the antigen (bait protein) and co-immunoprecipitate any interacting proteins (prey proteins). Traditional co-IP methods that use Protein A or G result in co-elution of the antibody heavy and light chains that may co-migrate with relevant bands, masking important results. The Pierce Co-IP Kit resolves this issue by covalently coupling antibodies onto an amine-reactive resin. The kit includes optimized buffers for protein binding and recovery, reagents to perform control experiments and efficient spin columns and collection tubes, which shorten the protocol and minimize handling and mixing.

Highlights:

• Works for any purified antibody – perform co-IP experiments using any purified antibody regardless of species or Ig class (chicken IgY, human IgE, mouse IgG1, IgM, etc.); no dependence on Protein A and Protein G affinity binding
• No antibody interference – covalent attachment method and non-reducing elution system yields pure co-IP products without contamination by the IP antibody
• Reusable antibody beads – covalent antibody immobilization allows prepared IP resin to be reused and provides potential savings of costly antibody
• Complete Kit – all reagents for antibody immobilization and many IP experiments, including binding and wash buffers, elution buffer and convenient microcentrifuge spin columns to make resin manipulations easier and more efficient.
• Control beads included – underivatized agarose beads provided for use as negative control for nonspecific binding
• Versatile – co-IP method is compatible with any physiolgical (non-denaturing) protein sample buffer that is compatible with the specific antibody-antigen and protein interaction complex.

Additional Information:

Co-immunoprecipitation (Co-IP) is a popular in-vitro method for discovering protein interactions. The Pierce Co-Immunoprecipitation Kit is configured to provide the essential tools to effectively perform Co-IP experiments in a manner that yields pure IP and co-IP products free of troublesome IP antibody. This improvement on the traditional co-IP technique is achieved by replacing Protein A/G agarose beads with AminoLink Plus Resin to which pure IP antibodies can be directly and permanently conjugated. Gentle (non-reducing, non-denaturing) elution buffer allows dissociation of the IP targets while retaining the immobilized antibody on the agarose beads. The result is a pure product that can be analyzed by Western blotting (or even sequenced) without interference from antibody.

Make leak-proof antibody beads. The Co-Immunoprecipitation Kit involves direct immobilization of purified antibody to aldehyde-activated beaded agarose (AminoLink Plus Coupling Resin). See details of coupling chemistry.

In addition to eliminating IP antibody contamination, the antibody-conjugation method integral to the Pierce Co-IP Kit avoids non-specific binding interactions and leaching contamination that can result from traditional Protein A and Protein G methods. Although the conjugation method requires that the starting antibody be in an amine-free buffer without BSA, gelatin or other storage protein stabilizers, the fact that it does not depend on Protein A or Protein G means that any antibody species or class can be used for IP (e.g., IgM and chicken IgY). Indeed any purified protein can be immobilized for use in affinity purification of binding partners.

Easy Co-Immunoprecipitation of p53 and MDM2 proteins. Purified mouse anti-MDM2 was coupled to AminoLink Plus Coupling Resin. Luciferase, MDM2 and p53 were translated and 35S-labeled. In vitro translated p53 (5µl) and MDM2 (5µl) were combined and incubated at 30°C for 30 minutes. Co-IP was performed at 4°C for 2 hours with 60µl anti-MDM2 antibody-coupled resin. Luciferase was used as a negative control protein to incubate with MDM2. Eluted proteins were resolved using 4-20% SDS-PAGE and visualized by autoradiography.

A.	B.	C.
Co-immunoprecipitation of transferrin and transferrin receptor protein (CD71) from serum using the Pierce Co-IP Kit. Samples were co-immunoprecipitated with anti-CD71 antibody immobilized to agarose beads according to the default kit procedure. Upon elution the samples were separated by 4-12% SDS-PAGE. One gel (A) stained for total protein with GelCode Blue Stain Reagent (#24590). Two gels (B and C) were transferred to nitrocellulose for Western blotting. Western blot (B) was probed with 500 ng/ml anti-CD71 antibody followed by 4 ng/ml Goat anti-Mouse-HRP and SuperSignal West Dura Chemiluminescent Substrate (#34075). Western blot (C) was probed with 100 ng/ml anti-human transferrin antibody followed by 4 ng/ml Rabbit anti-Chicken-HRP and SuperSignal West Dura Chemiluminescent Substrate. Lane 1. Chemiluminescent MW Marker (#26651), Lane 2. Agarose bead control, Lane 3. 50 ng purified transferrin, Lane 4. the Co-IP sample, and Lane 5. human transferrin receptor containing serum (diluted 1:10).

Related Products:
Other Immunoprecipitation Kits (guide to other methods)
Pierce IP Lysis Buffer
AminoLink Plus Immobilization Kit
Pierce Spin Columns
HA Tag IP/Co-IP Kit
c-Myc Tag IP/Co-IP Kit

References:

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3. Howell, et al. (2006). An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins. Proteomics 6:2050-2069.
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5. Rey, P., et al. (2005). Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses. The Plant Journal 41:31-42.
6. Rathmell, W.K., et al. (2004). In vitro and In vivo models analyzing von Hippel-Lindau disease-specific mutations. Cancer Research 64:8595-8603.