Precautions for Immunoprecipitation and Derivatization Experiments

Immunoprecipitation (IP) is a method developed by using the specific binding of antigen protein and antibody as well as the phenomenon that the "Protein A/G" of bacterial protein specifically binds to the Fc fragment of antibody (immunoglobulin). At present, protein A/G is commonly used to bind to argarose beads in advance, so that after reacting with the solution containing antigen and antibody, protein A/G on beads can achieve the purpose of adsorbing antigen. Through low-speed centrifugation, the target antigen can be separated from other antigens from the solution containing the target antigen.

Co-immunoprecipitation (Co-IP) is a classical method used to study protein interactions based on the specific action between antibodies and antigens and is an effective method to determine whether two proteins interact under physiological conditions. The principle is that many protein-protein interactions present in intact cells are preserved when the cells are lysed under non-denaturing conditions. If X is immunoprecipitated with an antibody to protein X, protein Y bound to X in vivo can also be precipitated. This approach is commonly used to determine whether two proteins of interest bind in vivo; it can also be used to identify new acting proteins for a specific protein.

Chromatin immunoprecipitation (ChIP) is an important tool to study the interaction between DNA and proteins in vivo. Its basic principle is to cross-link the intracellular protein and DNA under physiological conditions, break it into small chromatin fragments within a certain length range by ultrasound, then precipitate this complex through the target protein-specific antibody to be studied, specifically enrich the DNA fragments bound by the target protein, and know which genes the target protein interacts with by PCR detection of the target fragment.

Key points to be paid attention to in the experimental operation

Immunoprecipitation: Since the purpose of immunoprecipitation is mainly to capture the target protein through the interaction between antibody and target protein, the key point to be paid attention to in the experiment is the effect of the interaction between antibody and target protein. It is mainly affected by two factors: the binding ability of antibody, the target protein and the contactability of antibody. So the key factors in immunoprecipitation experiments are the selection of antibodies as well as the formulation in the lysis buffer. For the former, the action epitope targeted by the antibody must be on the surface of the target protein, and the requirement for antibody binding force is much higher than that for immunoblotting or immunofluorescence; for the latter, the main consideration is whether the detergent component in the buffer can effectively release the target protein from the local localization of the cell (such as located in some organelles) without disrupting the natural conformation of the target protein (without affecting the interaction effect of the target protein and the antibody).

Co-immunoprecipitation: The purpose of co-immunoprecipitation is to capture the target protein complex through the interaction between antibody and target protein, and finally detect the interaction between the components in the target protein complex. Therefore, in addition to paying attention to the effect of the interaction between antibody and target protein, attention should also be paid to the integrity of the target protein complex compared with immunoprecipitation. The main consideration of the latter is whether the detergent components in the buffer can effectively release the target protein complex into the soluble phase without destroying the interaction between the components in the target protein complex. Therefore, in addition to the selection of antibodies, the composition and concentration of detergents in the lysis buffer formulation are essential for the success of co-immunoprecipitation experiments, and their selection is stricter than that of immunoprecipitation experiments.

Chromatin immunoprecipitation: The main purpose of chromatin co-immunoprecipitation is to capture the DNA fragment interacting with the target protein through the interaction between antibody and target protein, and finally detect the interaction between target protein and DNA fragment. Therefore, the key points to be noted in the experiment are different from the former two (immunoprecipitation and co-immunoprecipitation). Because the maintenance of protein-DNA complexes in experiments depends on the cross-linking effect of paraformaldehyde, many surface epitopes of the target protein are also lost during cross-linking. Therefore, in addition to the slight differences in the subsequent basic operation and immunoprecipitation and co-immunoprecipitation, it is additionally important to note that the requirements for antibodies are much higher than those for immunoprecipitation and co-immunoprecipitation, and relatively speaking, the composition of lysis buffer is not a key factor.

Collected by Profacgen, an insect cell protein production provider.