| Immunoassay Wikipedia 6.0 |
| 6.0 Immunoprecipitation (IP) |
 6.1 Description
6.2 Protein-Protein IP
6.3 Protein-DNA (Chromatin) IP
6.4 Application
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| 6.0 Immunoprecipitation (IP) |
| Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. (Top) |
| 6.1 Description |
| In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate. The choice of agarose conjugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen-antibody system. (Top) |
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| Figure 1. Schematic of Immunoprecipitation Procedure. |
| An antibody added to a mixture of protein or cell lysate. Antibody-antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads. Upon centrifugation, the antibody-antigen complex is brought down in the pellet. Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Proteins captured by immunoprecipitation can be analyzed by SDS-PAGE or Western blot. |
| Immunoprecipitation followed by SDS-PAGE and immunoblotting is routinely used in a variety of applications: to determine the molecular weights of protein antigens, to study protein/protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation. (Top) |
| 6.2 Protein-Protein IP |
| Co-Immunoprecipitation (Co-IP) is a powerful method used to study protein/protein interactions. In Co-IP, one antibody is used to immunoprecipitate a target antigen and also co-precipitate any bound interacting proteins within a sample. This complex is then detected by Western blot using a second antibody targeted against one of the bound interacting proteins. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with a radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]-borohydride reduction to label carbohydrate). Having a radioactive antigen is not required but interpretation of data is simplified since the antigen, and not the antibody, is radiolabeled. Analysis of the immunoprecipitate is usually by electrophoresis although other techniques can be used. The choice of immobilized antibody binding protein depends upon the species that the antibody was raised in. Protein A binds well to rabbit, cat, human, pig and guinea pig IgG as well as mouse IgG2a and IgG2b. Protein G binds strongly to IgG from cow, goat, sheep, cow, horse, rabbit and guinea pig and to mouse IgG1 and IgG3. Protein G can also bind bovine serum albumin (BSA). Thus, BSA should be added to buffers used with Protein G. Alternatively, recombinant Protein G without BSA binding sites can be used (Protein G Plus from Oncogene Science). (Top) |
| 6.3 Protein-DNA (Chromatin) IP |
Traditional methods for performing Co-IP are optimal for studying DNA-binding protein complexes as the complexes are often disrupted during the extraction process. In addition, many unstable protein complexes can be affected by the salt and detergent composition of the buffers used in the immunoprecipitation process, which also complicates their analysis. Protein-DNA Co-IP has specific reagents for extraction and immunoprecipitation, which helps to maintain nuclear protein complexes, providing the best results.
Generally, nuclear extracts are prepared by collecting cells in ice-cold PBS with Phosphatase Inhibitors. Then, the cells are resuspended in Hypotonic Buffer to swell the cell membrane and make it fragile. Addition of detergent causes leakage of the cytoplasmic proteins into the supernatant. After collection of the cytoplasmic fraction, the nuclei are lysed and the nuclear proteins are recovered in a low-salt buffer in the presence of the Protease Inhibitor Cocktail and PMSF. This is followed by the addition of an Enzymatic Shearing Cocktail. The use of low-salt buffers protects protein complexes in the nucleus; DNA digestion allows a gentle release of un-dissociated protein complexes from the DNA. After the protein complexes are collected, an immunoprecipitation reaction is carried out to detect the bound proteins. Two different immunoprecipitation buffers with either a low or high stringency starting composition are provided. In addition, detergent and salt are provided separately to enable you to vary the salt and detergent concentrations. The addition of salt and detergent is ideal for use with robust protein/protein interactions because such conditions reduce background. However, as unstable protein complexes may not withstand high stringencies, variation in methods may be applied to enable high stringency but enough preservation of natural condition required by each complex.
Chromatin immunoprecipitation (ChIP) involves the immunoprecipitation of protein/DNA complexes that have been stabilized via cross-linking. Traditionally, transcription factor and gene promoter activity has been analyzed using reporter gene assays, EMSA, Western blot and DNA microarrays. Although these methods have led to significant advances in the scientific understanding of transcription, they cannot be used to demonstrate that a particular protein is bound to a specific, native DNA sequence in living cells. ChIP offers a versatile solution by combining the specificity of immunoprecipitation, the sensitivity of PCR and the screening power of array profiling, all in a single assay. However, it can also be technically challenging and difficult to validate without well-proven reagents. The better method and reagent kit are developed and optimized for this application. Figure 2 depicts the general process of ChIP. |
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| Figure 2. Schematic of Chromatin Immunoprecipitation. |
| In the ChIP method, intact cells are fixed using formaldehyde, which cross-links and therefore preserves protein/DNA interactions. DNA is then sonicated into small uniform fragments and the DNA/protein complexes are immunoprecipitated using an antibody directed against the DNA-binding protein of interest. Following immunoprecipitation, cross-linking is reversed, proteins are removed by Proteinase K treatment and the DNA is rapidly cleaned up using the included DNA purification columns. The DNA is then screened to determine which genes were bound by the protein of interest. The versatility of ChIP means that screening can be done using generalized hybridization or a more targeted PCR-based approach. (Top) |
| 6.4 Application |
| Immunoprecipitation or co-immunoprecipitation can be used for many purposes. Among these are: |
- Determination of the molecular weight and isoelectric point of immunoprecipitated proteins by one-dimensional or two-dimensional SDS-PAGE.
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- Verification that an antigen of interest is synthesized by a specific tissue (i.e., that radiolabeled protein can be identified in tissues or cells cultured with radiolabeled precursors).
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- Determination of whether a protein contains carbohydrate residues by evaluating whether immunoprecipitated antigen from cells cultured with radioactive monosaccharides is radiolabeled.
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- Characterization of the type of carbohydrate present on glycoproteins - evaluate incorporation of different radiolabeled monosaccharides into immunoprecipitated protein during cell culture and test whether inhibitors of glycosylation alter the molecular weight of immunoprecipitated protein.
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- Determination of precursor-product relationships by performing pulse-chase labeling followed by immunoprecipitation.
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- Quantification of synthesis rates of proteins in culture by determining the quantity of immunoprecipitated, radiolabeled protein.
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- Study of protein complex and interactions, including pulling down the complexes of protein-protein, protein-DNA or protein-RNA, etc. (Top)
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