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 To use the CNRU Mass Spectrometry Core Facility for your research you must first get started.
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IMMUNOPRECIPITATION AND WESTERN BLOTTINGImmunoprecipitation (IP) is one of the most widely used ways to measure the molecular weights of protein antigens
study protein/protein interactions
monitor specific enzymatic activity
track protein post-translational modifications
determine the presence and quantity of proteins
detect rare proteins which are concentrated up to 10,000-fold by the process
Immunoprecipitation involves incubating a lysate with an antibody that binds to a specific protein or peptide motif, then isolating the antibody-bound complexes using Protein G or Protein A attached to beads or resin. The beads are either washed stringently to disrupt any non-covalent interactions or washed mildly to retain interacting partners, then the samples are typically boiled in SDS and proteins are separated using gel electrophoresis. Once the proteins are separated by size, they are transferred to a nitrocellulose membrane. At the CNRU Mass Spectrometry Core Facility, we utilize a BioRad Criterion Blotter to transfer up to 2 gels at a time (in less than one hour). This provides a protein pattern on the membrane that should be identical to that on the gel. The nitrocellulose membrane is then incubated with an antibody specific to the protein or motif of interest (the primary antibody). The membrane is subsequently incubated with a secondary antibody that binds to the primary and is conjugated to some sort of reporting enzyme that can be visualized when the membrane is washed in an enzyme reaction mixture. In general, the enzymes will emit a flash of light that can be detected using an X-ray film. The blotting and detection is termed Western Blot analysis. Western blot analysis is typically used to detect one protein in a mixture of proteins and provide information on the relative amounts of the protein under different conditions. Please contact the CNRU Mass Spectrometry Core Facility for more information if you would like us to perform immunoprecipitations and/or Western blot analysis for you. |
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