What is immunprecipitation used for?
A Immunoprecipitation (IP, also known as co-immunoprecipitation) is a molecular biological method for the detection of protein-protein interactions. The protein interaction is demonstrated in an IP in vitroby using an antibody to precipitate a certain protein and its interaction partner from a protein mixture. The precipitated protein and its interaction partners are detected in the Western blot.
The protein mixture can be a homogenate of a tissue or cells from the cell culture. The cell culture also makes it possible to overexpress the partners of the suspected interaction, i.e. these proteins are increasingly formed. The interaction partner should still be bound to the other in this situation. After cells or tissue have been disrupted, an antibody is now added which specifically binds to one of the proteins. This antibody is then used to extract the protein and its interaction partner. The specific properties of so-called protein A and / or protein G, which is a component of the cell wall of certain streptococcal strains, are used here. Protein A and G bind with high specificity to the Fc region of most mammalian immunoglobulins. These proteins are then coated with beads (so-called beads, e.g. from Sepharose) in order to bind the antibody-protein complexes to itself in such an immunoprecipitation. The complexes are now usually isolated by centrifugation and several washing steps in order to remove unspecific proteins. The proteins are detached from the beads by denaturation and detection is carried out using a Western blot.
Advantages and disadvantages
The IP is used in molecular biology as an alternative proof of an interaction, e.g. B. after a yeast two-hybrid screen. It enables the investigation of protein-protein interactions in at least in vivo-like conditions, d. H. in the milieu of a cell and with post-translational modifications occurring in eukaryotes such as glycosylation (attachment of sugar chains), palmitoylation (attachment of fatty acids) or folding by chaperones.
However, it is possible that proteins change or are broken down when the cells are broken open. The success of IP also depends to a large extent on the binding of the antibody. Thus, false negative results can easily be produced (false negative), which can only be remedied by repeated test series with changed conditions. On the other hand, some proteins also bind directly to the beads or to the surface of the reaction vessels. These can simulate a non-existent interaction (false positive), which can only be remedied with additional controls.
Furthermore, it is possible that two proteins interact in the IP experiment, but do not occur simultaneously in the cell cycle, in the cell organelle or in the cell type and therefore cannot be actual interaction partners.
For the reasons given, the interpretation of IP results must be done with caution. Positive interactions always have to be verified with additional techniques from molecular biology, such as the yeast two-hybrid system or FRET. The IP experiment still provides information about the possible interaction of two proteins, but no information about how this interaction takes place. For this, more detailed studies of the structure of the proteins involved are necessary.
Category: Biochemical Detection Reaction
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