Immunoprecipitation (IP) is a technique for isolating specific proteins from solution by using antibodies capable of binding to proteins, and is one of the most widely used methods for antigen purification and detection. Antibody-protein complexes are removed from solution by the addition of antibody-bound proteins (e.g., Protein A or Protein G conjugated to agarose slurry or magnetic beads). Immunoprecipitation uses antigen-specific antibodies to isolate target antigens and analyzes the amount and size of antibody-reactive antigens in the complex-protein mixture using SDS-PAGE. Immunoprecipitation allows researchers to measure the molecular weight and quantity of a given protein in a protein mixture, identify protein activation states and post-translational protein modifications, and capture protein binding partners. Immunoprecipitation assays can detect the interaction of target proteins with other proteins or nucleic acids, among which Co-immunoprecipitation (Co-IP) assays commonly used for target protein detection.
Co-IP is a powerful technique widely used for the discovery and detection of protein-protein interactions. And it can be used to detect the interaction between two known proteins, or use a known protein to find an unknown protein that interacts with it. Compared to other molecular interaction assays (GST pull down, etc.), the advantage of Co-IP is that protein binding is done intracellularly, reflecting protein interactions in natural state with more realistic and reliable.
With the deepening of protein research, people combine immunoprecipitation method with other methods to derive many more complicated techniques, which make its application range quite extensive. Co-IP technology combined with methods such as western blotting or mass spectrometry is used to determine the binding situation of bait protein-target protein in the natural state, and to identify new acting molecules of specific proteins. Co-IP experiments can also be applied to the enrichment and concentration of low-abundance proteins.
If protein X is specifically precipitated with an antibody (anti-X), then proteins such as Y or Z, which are bound to X in vivo, can also be co-precipitated. Subsequently, the presence of protein Y in the precipitate was detected by western blot (WB).
Determining whether two target proteins bind in vivo
Identify new interacting molecules of a specific protein
Isolation of interacting protein complexes in their native state
Depending on your needs, Creative Proteomics offers Co-IP services to study protein-protein interactions using highly sensitive and specific immunoprecipitation-related reagents. In follow-up experiments, many methods such as west blot and mass spectrometry can be used to help customers analyze known and unknown interactors.
Collect protein samples—precipitate bait protein with bait protein antibody—separate protein by SDS-PAGE—detect target protein by WB—mass spectrometry (optional).
Diagrammatic representation of the co-immunoprecipitation (co-IP) procedure (Lin, J.S.; Lai, E.M. 2017)
CoIP-WB is the combination of co-immunoprecipitation and immunoblotting technology, which can verify whether the bait protein binds to the interacting protein, and then determine a certain signaling pathway through a series of functional experiments, and identify and verify the related proteins of the pathway.
CoIP-MS, the combination of co-immunoprecipitation and mass spectrometry, can use mass spectrometry to identify other members of this protein complex, and efficiently screen and identify unknown interacting proteins. The LC-MS/MS technique is used to identify and analyze the protein captured by Co-IP, which has higher sensitivity and almost 100% reliability, and is also the most commonly used method.
Integrated immunoprecipitation and mass spectrometry operations.
Provide world class quality labeled antibodies, affinity immunogels/magnetic beads.
Technical staff with years of experience in IP/CoIP operations to provide efficient, professional and fast service.
High quality LC-MS/MS equipment to provide highly reliable mass spectrometry results.
Sample type: Cell, tissue or RNA.
a) Cells: 2×107
b) Tissue: 500 mg