![]() Antibody-binding proteins affect IP methodology in two ways. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.īoth prokaryotic and eukaryotic systems have antibody-binding proteins. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The constant domain identifies the type of antibody and dictates function in vivo. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Please click here to view a larger version of this figure.Īntibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. ![]() Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. The isolated protein is then analyzed by Western blot. In pull down step, the target protein is pulled down using a protein-specific antibody. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. Immunoprecipitation is the isolation of a specific protein using an antibody. protein, DNA, RNA) (3, 4, 5).įigure 1: Overview of Immunoprecipitation Procedure. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein's interacting partners (i.e. The combination of IP with subsequent techniques is a powerful analysis tool. An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). A necessary step after immunoprecipitation is verification of purification. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. myc or flag) attached to them to simplify subsequent purification. Most recombinant proteins have an epitope tag (i.e. The target protein might be endogenous or recombinant. The word "immuno" refers to the use of an antibody while the word "precipitation" refers to pulling down a specific substance from a solution. The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. First conceived in 1984, it was refined in 1988 (1, 2). Immunoprecipitation (IP, also known as a 'pull-down' assay) is a widely used technique that has applications in a variety of fields. Webb 1ġ Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
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