Immunoprecipitation
An immunoprecipitation ( IP , English immunoprecipitation ) is a biochemical method in which an antigen is concentrated from a solution in a pulldown assay using an antibody .
principle
The antigen to be purified is mostly a biopolymer , e.g. B. a protein , peptide , polysaccharide or a nucleic acid . For proteins, binding of an antibody to protein tags is often used, e.g. B. the TAP day . The purification of nucleic acids by immunoprecipitation is described for DNA under chromatin immunoprecipitation , also under ChIP-on-Chip and ChIP-Seq , as well as for RNA under RIP-Seq ( iCLIP , PAR-CLIP and CLIP-Seq ) and RIP-Chip .
As a rule, the antibody is coupled in vitro to a solid stationary phase ( cross-linked agarose or dextran beads ) and, through its affinity, binds a specific antigen in a solution, for example in a tissue lysate clarified by filtration or centrifugation . A certain antigen and its interaction partners (coprecipitates) are precipitated from a mixture . This means that immunoprecipitation is also suitable for the detection of protein-protein interactions, since whole protein complexes can be precipitated with this method. In order to reduce unspecific interactions between undesired molecules, the binding sites of the stationary phase are saturated with sufficient quantities of the target protein so that binding molecules are bound as specifically as possible.
A precipitated protein and its interaction partners can then be detected using various methods, for example with immunoconjugates in a Western blot , peptide mass fingerprint or by prior labeling with radioimmunoconjugates .
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. H. these proteins are increasingly formed. The interaction partner should still be bound in this situation. After cells or tissue have been disrupted, antibodies are added that specifically bind to one of the proteins. These antibodies are then used to sediment the protein and its interaction partners from the solution by centrifugation or MACS . Several washing steps serve to remove non-specifically bound proteins. The proteins are loosened from the beads by denaturation and detection is usually carried out using a Western blot .
As a rule, use is made of the specific properties of so-called protein A , which comes from the cell wall of the bacterium Staphylococcus aureus , and / or protein G , which is a component of the cell wall of certain streptococci strains. Protein A and G bind with high specificity to the Fc region of most mammalian - immunoglobulins . These proteins are then coated with beads (e.g. beads made from Sepharose or magnetic microparticles ) in order to bind the antibody-protein complexes to them in such an immunoprecipitation.
Quantitative immunoprecipitation
Precipitated or soluble antigen-antibody immune complexes cloud a solution. If purified antigen standards are used, conclusions can be drawn about the antigen concentration in the sample by measuring the turbidity by immunophelometry .
Advantages and disadvantages
The IP is used in the course of protein purification and protein characterization as an alternative evidence 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, i. H. in the milieu of a cell and with post-translational modifications that occur 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 as the cells break open . Another problem with the method is a lack of purity of the binding protein in immunoprecipitation, which can lead to false positive results. Furthermore, the results of the immunoprecipitation are partly dependent on the specific binding of the antibody, heterophile antibodies can non-specifically precipitate undesired proteins. On the other hand, some proteins also bind directly to the beads or to the surface of the reaction vessels. These bound molecules can simulate a non-existent interaction (false positive), which can only be eliminated through additional control attempts. 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. Since false negative results can also be produced under suboptimal environmental conditions , repeated test series with changed buffer conditions serve to reduce false negative results.
For the reasons mentioned, the interpretation of IP results must be done with caution. Positive interactions always have to be verified with additional techniques, such as the yeast two-hybrid system or FRET . Immunoprecipitation 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.
Individual evidence
- ↑ B. Kaboord, M. Perr: Isolation of proteins and protein complexes by immunoprecipitation. In: Methods Mol Biol. (2008), Volume 424, pp. 349-364. doi : 10.1007 / 978-1-60327-064-9_27 . PMID 18369874 .
- ↑ C. Dickson: Protein techniques: immunoprecipitation, in vitro kinase assays, and Western blotting. In: Methods Mol Biol. (2008), Volume 461, pp. 735-744. doi : 10.1007 / 978-1-60327-483-8_53 . PMID 19030838 .
- ↑ Bonifacino, JS, Dell'Angelica, EC and Springer, TA 2001. Immunoprecipitation. Current Protocols in Molecular Biology. 10.16.1-10.16.29.
- ^ Ian Rosenberg: Protein analysis and purification: benchtop techniques . Springer, 2005, ISBN 978-0-8176-4340-9 , p. 520.
- ^ SC Masters: Co-immunoprecipitation from transfected cells. In: Methods Mol Biol. (2004), Volume 261, pp. 337-350. PMID 15064468 .
- ↑ Christine Schütt, Barbara Bröker: Basic knowledge of immunology, 2nd edition, Springer, 2009, ISBN 9783827420275