Method of the Month: Immunoprecipitation
September 2024
Background
In the 1950s, scientists became increasingly interested in studying proteins, specifically, how proteins interact with the immune system and the genome of a cell. To understand basic cell functions, it was necessary to understand how proteins interact with one another. To do that, it was important to isolate proteins from their environment. First described by Dr. Alfred Nisonoff and his colleagues, immunoprecipitation is one of the first methods of protein separation using a basic facet of the immune system: antibodies.
Method
Immunoprecipitation is a protein separation method using antibodies to study protein-protein, DNA-protein, or RNA-protein interactions. This method is characterized by the following steps: cell lysis/protein separation, antibody binding, cross-linking antibodies, and protein extraction.
Cell Lysis is the process of breaking open cells collected from tissue to release its internal content including proteins and genetic material. There are a few ways to lyse a cell: mechanical, chemical, or enzymatic. Mechanical lysis might include using tiny beads to break down cells or freezing cells so they burst. Chemical lysis uses a hypotonic solution to burst the cells (osmotic lysis) or buffers to degrade cell walls. Enzymatic lysis involves using a variety of enzymes that digest or dissociate cell walls. The point of this step is simply to free the material inside a cell, so the best lysing method will be based on the target protein and its efficiency for the next step: Antibody binding.
Antibody binding is the specific interaction between an antibody and its antigen. Antibodies are proteins structurally designed to recognize a specific target protein with its antigen-binding site. Antibodies can be designed for antigens or any other target protein. Antigens, on the other hand, are immunocompromising proteins with the epitope, the specific antibody’s target structure. Once the cell is lysed from the first step, proteins from inside the cell are released. The target protein becomes the antigen. Into the complex mixture, scientists add the antibody which is specific to the protein they are targeting. The antigen-antibody binding allows scientists to recognize the target protein so they can move on to the next step: Cross-Linking Antibodies.
Cross-linking antibodies is the process of stabilizing antigen-antibody complexes by covalently linking them together with chemical cross-linkers. Stabilizing is necessary for the processes downstream of this method. Cross-linkers are chemicals that bind the antigen-antibody or protein-antibody complexes. The most common cross-linker for immunoprecipitation is Dimethyl pimelimidate (DMP). DMP is an amine-reactive cross-linker often used to cross-link antibodies to Protein A or G agarose or magnetic beads. This forms an even larger complex that makes isolating the target protein much easier. Once the complex forms, the mixture is centrifuged to separate the heavier antibody complexes with the beads from the rest of the previously lysed content.
Once these steps are complete, the newly isolated antigen-antibody complex can be tagged for protein visualization methods like western blotting or spectrometry.
Application
Immunoprecipitation is the first step to many recent assays that require isolated proteins for research. Clinical research on diseased tissue or genetic diseases that affect protein function will require isolated proteins for study, and Immunoprecipitation is the foundation of that research.
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