Isolation of proteins and protein complexes by immunoprecipitation.
Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.
Kaboord
Barbara
B
Pierce Biotechnology Inc., Rockford, Illinois, USA.
Perr
Maria
M
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology
D000906:Antibodies; C029152:G-substrate; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D013205:Staphylococcal Protein A