Protein Structure and Function Lecture 4 Notes: Protein-Protein Interactions Part 2 Immunoprecipitation, Co-Immunoprecipitation and Pull-Down Assays - Immunoprecipitation (IP) is a popular technique used for protein purification. It relies on PPI to capture the protein of interest. Incubation with Antibody-coupled Resin Cell Lysate or Protein Mixture - An antibody-coupled resin containing an antibody against a specific target antigen forms an immune complex with its target thus separating this (it is immobilised) from the rest of the unrelated proteins so it can be eluted/purified. CD Spin and wash Blute Coupled Antibody Antigen - This interaction can be between the whole Immunoprecipittaion (IP): - populartechnique for protein purification Analyze Incubation with Antibody-coupled Resin Cell Lysate or Protein Mixture antigen and antibody-against-antigen or between the tagged protein and antibody of antigen. Co-immunoprecipitation (co-IP) is a popular technique for protein interaction discovery. relies on PPI to capture/purify the antigen of interest as well as to identify/capture a protein which interacts with the antigen (i.e. two PPIs). The analysis of this solution depends on breakdown of the contents. It Spin and wash Eluto < Coupled Antibody Antigen Protein Interacting with Antigen Co-immunoprecipitation (co-IP): Analyze - A pull-down assay is used for the analysis of PPI. They are a form of affinity purification. A bait protein is used instead of an antibody (otherwise is similar to co-IP). This is convenient for affinity-tagged bait proteins (e.g. GST-, poly-His- or Btn-tagged proteins). - The bait protein is immobilised with an affinity ligand (e.g. Glutathione) attached to an agarose bead. 'Prey' protein is added to the mixture so that it can bind to the bait and can be captured. The PPI complex is displaced (elution) by another molecule for analysis. - IP, co-IP and pull-down assays rely on immobilised affinity capture reagents. This makes purification and detection much easier. This could be in the form of: (a) Membranes (e.g. NC, PVDF), arrays and microarrays (e.g. glass) (b) Fluorescent beads, microbeads or quantum dots (for sensitive detection) (c) Porous beads (e.g. agarose, sepharose) and micro-/nanoparticles (analytical) (d) Gold (or other) nanoparticles (for high surface/volume ratio or visual detection) - Antibodies (or bait proteins) can be directly or indirectly coupled via PPIs to solid support. Direct coupling is to a protein A/G (binding to Fc), GST (binding to glutathione labelled IgG) or Avidin/Streptavidin (bind biotinylated IgG). This method is simple (i.e. requires mixing), reversible (can regenerate) and oriented, however it can leak IgG or bait protein. Direct (covalent) binding (e.g. though NH2- or SH- groups) does not leak because it is irreversible (cannot regenerate) however it is also not oriented in most cases and depends on chemical compatibility (i.e. buffers).
- One of the most commonly used covalent coupling affinity ligands is n-hydroxysuccinimide (NHS) ester in activated agarose resin. NHS reacts with primary amines (NH2) on proteins or other molecules to form stable amide linkages (common technique). - Direct coupling via sulfhydryl (SH-) groups may require the reduction of disulphides in solution or using immobilised reduction agents prior to immobilisation -