….proteins that interact with…
Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks.
Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks.
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In vitro Methods
Co-Immunoprecipitation (Co-IP) An immunoprecipitation (IP) experiment designed to affinity purify a bait protein antigen together with its binding partner using a specific antibody against the bait.
Cross-linking Reagents Strategies involve homo- or heterobifunctional reagents whose chemical cross-links may or may not be reversed. Nearest neighbors (suspected to interact)in vivo or in vitro can be trapped in their complexes for further study.
Far-Western Analysis Similar strategy to Western blotting with one key difference. The antibody probe in a typical Western blot detection is substituted with an appropriately labeled bait protein as the probe. Detection can be radioisotopic, chemiluminescent or colorimetric, depending on the probe label.
Label Transfer Involves a specialized cross-linking agent with several important features. These include heterobifunctionality for stepwise cross-linking, a detectable label and reversibility of the cross-link between binding partners. Upon reduction of the cross-linked complex a binding partner (prey protein) acquires the label from a bait protein that was first modified with the reagent. The label is typically used in the detection process to isolate or identify the unknown prey protein.
Protein Arrays/ Protein Chips Antibody-based or bait-based arrays that allow for screening and detection of specific interactions of proteins from complex mixtures. Primary applications include high-throughput assays of protein expression profiling, protein:protein interaction and enzyme activity. Most of the current protein chips are based on the binding between the capture proteins immobilized on a surface and the target proteins in the sample solution.
Protein Interaction Mapping Utilizes an “artificial protease” on a bait protein to initiate contact-dependent cleavages in the prey protein in the presence of specific reactants. The nonspecific cleavage fragments produced by the artificial protease can be analyzed to map the contact sites or interface of a known protein:protein interaction.
Pull-Down Assays An affinity chromatography method that involves using a tagged or labeled bait to create a specific affinity matrix that will enable binding and purification of a prey protein from a lysate sample or other protein-containing mixture.
Surface Plasmon Resonance Relates binding information to small changes in refractive indices of laser light reflected from gold surfaces to which a bait protein has been attached. Changes are proportional to the extent of binding. Special labels and sample purification are not necessary, and analysis occurs in real time.
NMR (Nuclear Magnetic Resonance) Method that can provide insights into the dynamic interaction of proteins in solution.
Mass Spectroscopy Used in concert with affinity-based methods, such as co-IPs, to isolate binding partners and complexes and identify the component proteins using standard mass spectral methods, e.g., MALDI-TOF and mass searching of bioinformatics databases.
X-ray Crystallography Crystallization of the interacting complex allows definition of the interaction structure.
Source: http://www.piercenet.com/Proteomics/browse.cfm?fldID=A6C04192-4535-4618-B372-98D97A7A21F8
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