Immunoprecipitation resins

Protein immunoprecipitation

Protein immunoprecipitation (IP) is a small-scale affinity purification technique that uses specific immobilized antibodies to isolate target antigens. Solid supports such as agarose resin and magnetic particles immobilize the antibodies used in IP. During IP, an antibody for the target protein is incubated with a cell extract that enables the antibody-protein binding. Protein A- or protein G-coupled agarose beads are then used to pull the antigen-antibody protein complex from samples like cell lysates to isolate target proteins.

Use an antibody with high binding affinity and specificity for the antigen of interest to ensure successful IP. Lysis buffers reduce protein denaturation and help release adequate amounts of proteins from samples. Protect the integrity of proteins of interest during cell lysis using protease inhibitors. A common application of IP is the separation of biomolecules from tissue or cell lysates to allow subsequent detection by immunoprecipitation assay techniques like western blotting. Launch your IP journey with the Immunoprecipitation Starter Pack from Cytiva.

Comparison between agarose and magnetic beads

Immunoprecipitation can be done with agarose beads or magnetic beads. Agarose-based resins are porous and mesh-like, allowing antibodies to diffuse and bind to their internal matrix. Their unique structure offers high binding capacity. Separating these sponge-like agarose beads from buffer and sample solutions is often done by centrifugation.

Magnetic beads can be used for protein immunoprecipitation as well as purification of other biomolecules, such as DNA. When using magnetic beads, high-power magnets localize the beads to the side of incubation tubes, allowing aspiration of cell lysates with minimal risk of aspirating undesired protein complexes. Cross-linking IP antibodies with protein G or A magnetic beads enables protein immunoprecipitation. Magnetic separation avoids centrifugation to help preserve antibody-antigen binding. Other advantages of magnetic beads include:

• High purity and reproducibility
• Ease of use
• Speed and automation
• High capacity and yield

Protein immunoprecipitation FAQs

Below are answers to some frequently asked questions about protein immunoprecipitation.

What is the purpose of immunoprecipitation?
The purpose of protein immunoprecipitation is to isolate proteins from complex mixtures to allow subsequent detection by assay techniques. IP is often used for analytical purposes or to screen ligands before larger preparative purification processes.

Where is immunoprecipitation used?
Common applications of immunoprecipitation are protein isolation, determination of concentration of protein levels, evaluation of protein post-translational modifications (PTMs), and identification of protein-protein interactions.

Can immunoprecipitation be used for DNA, RNA, or protein?
Yes, IP principles can be used to isolate DNA and RNA in addition to proteins.

How many types of immunoprecipitations are there?
Two very common classifications of immunoprecipitation methods are direct and indirect IP.

• Direct method = pre-immobilized antibody approach: In this method, the antibodies are attached to the beads in advance. The direct method gives you better control of the antibody binding and immobilizes the antibodies covalently to the beads. This way, you can make sure that the antibodies will not elute and interfere with the subsequent analysis.
• Indirect method = free antibody approach: In this method, antibodies are incubated with the antigen before beads are added. The indirect method is very easy to use, has fewer steps, and can be used if the antibodies do not interfere with following steps.