Immunoprecipitation

( Called IP, and immunoprecipitation ) Immunoprecipitation is a biochemical method in which in a pull-down assay using an antibody, an antigen is concentrated from a solution.

Principle

The antigen to be purified is typically a biopolymer, such as a protein, peptide, polysaccharide or a nucleic acid. In proteins often the binding of an antibody to protein tags is used, for example when the TAP tag. The purification of nucleic acids by immunoprecipitation is described in chromatin immunoprecipitation, even under ChIP -on-chip and ChIP -Seq, DNA -Seq and RIP ( iCLIP, PAR -CLIP and CLIP -Seq ) and RIP - chip for RNA.

In general, the antibodies in vitro in this case coupled to a solid stationary phase ( cross-linked agarose and dextran ), and binding of its affinity, a specific antigen in a solution, for example, in a tissue lysate clarified by filtration or centrifugation. A specific antigen is here with all its interaction partners ( coprecipitates ) thus precipitated from a mixture. As a result, the immunoprecipitation is also suitable for the detection of protein -protein interactions, since all protein complexes using this method can be precipitated. To reduce non-specific interactions of unwanted molecules, the binding sites of the stationary phase will be saturated with sufficient quantities of the target protein, such that binding molecules as possible are specifically bound.

A precipitated protein and its interaction partners can be detected in connection with different 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 allows the possibility to overexpress the putative interaction partners, ie these proteins are preferentially formed. The interaction partners should still be bound in this situation. After cells or tissues were broken, it is now added antibodies that specifically bind to one of the proteins. About this antibody the desired protein together with interaction partners from the solution by centrifugation or MACS is then sedimented. Several washing steps are used to remove non-specifically bound proteins. The proteins are released from the beads by denaturation, and the detection is carried out mostly on a Western blot.

Here, use is made in general to the specific properties of so-called Protein A, which is derived from the cell wall of the bacterium Staphylococcus aureus, and / or protein G, which is a component of the cell wall of certain Streptococcus strains. Protein A and G bind with high specificity to the Fc region of most mammalian immunoglobulin. These proteins are coated beads now (English beads, eg Sepharose or magnetic microparticles ) to bind in such immunoprecipitation, the antibody -protein complexes in itself.

Quantitative immunoprecipitation

Unusual or soluble antigen-antibody immune complexes tarnish a solution. If one uses purified antigen standards as can be concluded that the antigen concentration in the sample by measuring the turbidity by immunonephelometry.

Pros and Cons

Is used the IP in the course of protein purification and characterization as an alternative detection of an interaction, eg after a yeast two- hybrid screen. It allows the study of protein - protein interactions in vivo, at least in similar circumstances, that is, in the milieu of a cell and occurring in eukaryotes, post-translational modifications such as glycosylation ( attachment of sugar chains), palmitoylation ( appending fatty acids) or a convolution by chaperones.

But it is possible that change proteins by rupture of the cells, or be reduced. Another problem with the method is a lack of purity of the binding protein that can lead to false -positive results of immunoprecipitation. Furthermore, the results of the immunoprecipitation are dependent in part on the specific binding of the antibody, heterophilic antibodies can precipitate unwanted proteins non-specifically. On the other hand, some proteins also bind directly to the beads or to the surface of the reaction vessels. These bound molecules can a non-existent interaction pretend ( false positive), which can only be resolved by additional control experiments. Furthermore, it is possible that two proteins indeed interact in the IP experiment, but in the cell cycle, do not occur in the organelle or cell type at a time and therefore may not be actual interaction partners. Since under suboptimal environmental conditions false negative results can also be produced, used repeated series of experiments with modified buffer conditions a reduction of false-negative results.

For these reasons, the interpretation of IP results must be cautious. Positive interactions should always be verified with other techniques such as yeast two -hybrid system or FRET. Immunoprecipitation are indeed information about the possible interaction of two proteins, but no information about how this interaction takes place. These detailed studies of the structure of the proteins involved are necessary.