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Immunopr ecipitation & E LISA

Immunopr ecipitation & E LISA. Dimitri Woods. Immunoprecipitation. What is it?.

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Immunopr ecipitation & E LISA

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  1. Immunoprecipitation & ELISA Dimitri Woods

  2. Immunoprecipitation

  3. What is it? • Immunoprecipitation uses antibodiesspecific to a particular protein in an aqueous solution to remove those proteins. Theantibodies are precipitatedout of thesolution while an insoluble form of theantibody binding protein is added to thesolution.

  4. Types of Immunoprecipitation • There are 5 types of immunoprecipitation • Individual protein immunoprecipitation • This type of immunoprecipitation pertains to using the specific antibody for a known protein and isolating it out of the solution • Protein complex immunoprecipitation • Protein complex (Co-IP) refers to using a known protein’s (from a larger complex of proteins) antibody in order to pull out the other proteins from that complex to later identify • Chromatin immunoprecipitation • Chromatinimmunoprecipiation (ChIP) is used for finding and locating DNA proteinbinding sites on the genome for the protein of interest • RNA immunoprecipitation • RNA immunoprecipitation (RIP) is essentially the same as ChIP, but instead of DNA proteinbinding sites, it looks for RNA protein binding sites. • Tagged Proteins • This is not really a typeofimmunoprecipitation, but rather a method in immunoprecipiation. This is when we engineer a tag on either the 3’ or 5’ ends of astrand of DNA. The tag can be reused, and on different proteins each time.

  5. Method • Indirect • Protein specific antibodies are directly put into the protein solution. They freely float about and latch onto their targets. After a certain time period, beads coated in protein A/G is added to the mixture. The antibodies will then stick to the beads • Direct • The protein specific antibodies are isolated onto a substrate (i.e. superpamagnetic beads). The beads with bound antibodies are added to the mixture, and the antibodies attract the proteins onto the beads.

  6. ELISA Enzyme-Linked Immuno-Sorbent Assay

  7. Purpose • ELISA is used to find the antibodies and/or antigens in a particular sample.

  8. How does it work? • With an unknown amount of antigen, ELISA employs the specific antibody for the antigen binding to the antigen. The antibody is connected to an enzyme, which later, will be able to provide a sort of detectable signal

  9. Uses • Very good for detecting infectious diseases (i.e. West Nile Virus, HIV/AIDS) • Useful for determining allergies

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