Immunoprecipitation (IP), Co-immunoprecipitation (Co-IP) and Pull-down assays are approaches designed to study the presence, abundance, up regulation or down regulation, post translational modifications and interactions of proteins. Previously, the standard purification and detection methods were radio-immuno labeling assays (RIA). The use of radioactive isotopes to label antigens can still be found in use today, however, safety and regulatory concerns along with cost have led to the development of new methods free from this type of labeling. With advances in these new techniques, antigens are able to be purified directly from the lysate, resolved by SDS-PAGE and then detected by western blotting, ELISA or quantified by mass spectrometry with the equivalent sensitivity that was first seen in radio-immuno labeling assays.
Immunoprecipitation Assay The Immunoprecipitation assay (IP) is designed to detect and purify a specific protein from a homogenate, such as cell or tissue lysate. This method involves incubating an antibody specific to the protein of interest along with cell or tissue lysate to form an immune complex. This complex is then precipitated onto an immobilized support, typically agarose resin. Any protein not bound to the agarose resin is subsequently removed during the washes. Finally, protein is eluted from the beaded support through a series of washes and collected through centrifugation. The sample is analyzed by SDS-PAGE, often followed by Western blot detection.
Co-Immunoprecipitation Assay The Co-Immunoprecipitation assay (Co-IP) is based on the same methodology as immunoprecipitation in its ability to capture and purify an antigen of interest; however, Co-IP is focused on the additional molecules that are bound to the target protein by inherent interactions in the sample complex. These interacting proteins may be complex partners, structural proteins, co factors, or signaling molecules. The significance of the Co-IP assay is useful its identification of protein to protein interactions thereby elucidating signaling pathways.
FLAG® Affinity Gels
The FLAG® Expression System is a proven method to express, purify and detect recombinant fusion proteins. FLAG and 3xFLAG are useful in the study of protein-protein interactions. These small hydrophilic tags significantly improve detection and purification of recombinant fusion proteins when used with our highly specific and sensitive ANTI-FLAG antibodies.
Hemagglutinin (HA), is a commonly used peptide derived from the human influenza virus. This small peptide containing 9 amino acids, YPYDVPDYA, can be incorporated into an expression vector at the N-terminus, the C-terminus or internally. This fusion tag is popular given its lack of bioactivity and biodistribution of the recombinant protein. The HA-Tag is preferred when a small tag is required, a low metabolic load is necessary to maintain normal physiology of a cell, and when the target protein is intended for post purification crystallization.
A small peptide consisting of 11 amino acids, N-EQKLISEEDL-C, taken from the human c-Myc protein, can be incorporated into an expression vector at the N-terminus, the C-terminus, or internally. The Myc-Tag is ideal when a small tag is necessary to maintain protein function, a low metabolic load is required to maintain the physiology of a cell, and when then target protein will be used for post purification crystallography. A disadvantage of using the tag is that it may interfere with translocation into the secretory pathway. It is important to avoid fusing the tag directly behind the signal peptide of a secretory protein.
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