32.10: Immunoprecipitation
Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or protein-RNA interactions. It is an important technique for studying crucial cellular processes such as gene transcription, DNA replication, recombination and repair, cell cycle progression, and epigenetics. It is also helpful to identify the in vivo location of binding sites of various transcription factors, histones, and other regulatory proteins.
The major steps in ChIP include fixation, sonication, immunoprecipitation, and analysis. It involves cross-linking the target protein with the DNA using a fixing agent, such as formaldehyde, followed by sonication or enzymatic hydrolysis to obtain smaller chromatin fragments. The technique then utilizes high-affinity antibodies specific against the protein of interest to capture the DNA bound to the protein in an immunoprecipitation reaction. The cross-linking is then reversed using high heat, high salt concentration, and proteinase K to release the DNA from associated proteins. The DNA is further purified to prepare it for analysis.
Cross-linking Immunoprecipitation
Cross-linking immunoprecipitation, or CLIP, identifies the regions of protein binding sites on endogenous RNA by co-precipitating them in the active transcription phase. RNA molecules are cross-linked to proteins to hold them together tightly and prevent their degradation. The procedure for the breakdown and isolation of the complexes is similar to ChIP. This technique is used to study the interaction of RNA with RNA binding proteins and their modifications in various biological systems.
Limitations of IP
Though immunoprecipitation techniques tend to reduce the number of purification steps, it has certain limitations. The antibody binding recombinant bacterial proteins, proteins A or G, conjugated to agarose beads, and antibodies used in the method may undergo non-specific binding, introducing contaminants in the purified protein preparation. Also, the immobilization of antibodies on beads requires optimization and is time-consuming. The washing of the beads after the antigen-antibody complex is critical, but there is a chance of losing the target protein at this step.
