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  • Co-Immunoprecipitation (Co-IP) - Thermo Fisher Scientific

    Transféré par

    Shailendra Yadav
    8 vues6 pages
    co ip

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    Co-Immunoprecipitation (Co-IP) _ Thermo Fisher Scientific

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    8 vues6 pages

    Co-Immunoprecipitation (Co-IP) - Thermo Fisher Scientific

    Transféré par

    Shailendra Yadav
    co ip

    Droits d'auteur :

    © All Rights Reserved
    Téléchargez comme PDF ou lisez en ligne sur Scribd
    Signaler comme contenu inapproprié
    sur 6
    Home » Life Sciences » Protein Biology » Protein Bicloyy Learning Center » Protein Biology Resource Litrary » Pierce Protein Methods » Co-hnmunopreciaton cow) Co-immunoprecipitation (Co-IP) Co-immunoprecipitation (Co-IP) is a popular technique to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein, These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein. Immunoprecipitation (IP) vs. Co-immunoprecipitation (Co-IP) The topie of co-mmunoprecipiation (Co-IP} is best preceded by a review of immunopreciptation (IP) fo help frame an understanding of the princiolos involved. The description of IP methodology here is brit Immunoprecipitation (IP) Immunopreciptation is one of the most widely used methods for antigen detection and purification. The principle of an IP is very straightforward: an antibody (monoclonal or polyclonal) against a specific target protein forms an immune complex with that target in a sample, such as a coll lysate. The immune complex s then captured, or precipitated, on 2 beaded supporto which an antbody-binding protein is immobilized (such as Protein Aor G), an ‘any proteins not precipitated on the beads are washed away. Finally, the antigen (and antibody, iit s not covalently attached to the beads andior when using denaturing buffers) is eluted from the support and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), often followed by Wester blot detection to verity the identty of the antigen. Learn more + Overview of Pretein-Protin Interaction Analysis + Overew of Waster Bot Anaiyaie Call Lysate or Incubation with Protein Mixture ‘Antibody coupled Resin 2°" ey a eos os fm e a“ ® Ln) a DK coupes ansvoay A Antigen Co-Immnunoprecipitation (Co-IP) Corimmunoprecipitation is an extension of IP that is based on the potential of IP reactions to capture and purify the primary target (Le, the antigen) as well as other macromolecules that are bound to the target by native interactions in the sample solution. Therefore, whether or nat an experiment is calle ‘an IP or co-IP depends on wiethar the focus of the experiment isthe primary target (antigen) or secondary targels (interacting proteins). Call Lysate or Incubation with Protein Mixture Antibody-coupled Resin a4 4 e e& Coupled Antibody A Antigen ‘SD Protein interacting swith Antigen Co-IP Optimization Strategies While the co-IP methadalogy is straightforward, performing a co-IP reaction and identiying physiological protein-protein interactions can be dificult, because of the nature ofthe interaction, nonspecific binding to IP components and antibody contamination that may mask detection. The following ‘sections describe each aspect of the co-IP approach that can be optimized to imprave detection Complex Binding Because co-immunopreciplation depends so much on protein-protein interactions in order to detect the bound proteins, the abilly to maintain stable physiological interactions throughout the mechanical and chemical stresses of the incubation and washing steps is a erticl factor when performing a co: IP reaction. Therefore, low-affinity o transient protein-protein interactions may not be detected by co-IP unless the interaction can be stabilized, ‘A.key factor in maintaining complex formation throughout the steps required for co-IP isthe lysis and wash buffers. Many protein interactions wil main intaet afte lysis using standard non-denaturing isis buffers, as described inthe Immunopreciptation method in the Proteln Methods Library. Buffers wit low ionic strength (i, <120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100) are less ikely to disrupt protein-protein interactions; however, empirical testing may be required to determine the best buler formulation for a specific protein complex of interest. ‘Additionally, lysing cells by sonication or vortexing the lysate or bead-bound immune complexes during the wash steps should be avoided to prevent the

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