Immunoaffinity purifications

Immunochemical approaches allow for the purification of a protein from a complex mixture in a single chromatographic step. Since antibodies to the protein targeted for purification are required, immunoaffinity purification techniques are often used for the routine purification of a protein subsequent to its initial purification by traditional chromatographic approaches. Immunoaffinity purification of a protein involves the following general steps  

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T.M. Phillips and B.F. Dickens, Affinity and Immunoaffinity Purification Techniques, Eaton Publishing, Natick MA, 2000. ISBN 1881299228. 

Immunoaffinity purification of elF3 containing FLAG-tagged... 

Immunoaffinity purification of elF3 containing hemagglutinin-tagged elF3i/TIF34. (HA pull-down)... 

Recently, Vasilescu et al. demonstrated the use of formaldehyde to preserve protein interactions in vivo followed by immunoaffinity purification of a targeted complex, cross-link reversal via heating at 95°C, separation by SDS-PAGE, and identification of bands by LC-MS/MS.7 Tagwerker et al. utilized formaldehyde cross-linking in conjunction with a novel tag-based affinity purification method.36... 

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

Nakayasu, H., Nishikawa, M., Mizutani, H., Kimura, H., and Kuriyama, K. (1993) Immunoaffinity purification and characterization of gamma-aminobutyric acid (GABA)b receptor from bovine cerebral cortex../. Biol. Chem. 268, 8658-8664. 

Kaware M, Bronshtein A, Safi J, Van Emon JM, Chuang JC, Hock B, Kramer K, Altstein M (2006) Enzyme-linked immunoassay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples. J Agric Food Chem 54 6482-6492... 

Other common impurities, such as immunoglobulins and protein A, result from the immunoaffinity purification of recombinant proteins or MAbs.16 If affinity chromatography is used to purify an antigen, then an ELISA can be used to detect contaminating levels of MAbs leached from the column. An assay for the antibody needs to detect the antibody in the presence and absence of its specific antigen. 

Lucas, C., C. Nelson, M.L. Peterson, S. Frie, D. Vetterlein, T. Gregory, and A.B. Chen (1988). Enzyme-linked immunosorbent assays (ELISAs) for the determination of contaminants resulting from the immunoaffinity purification of recombinant proteins. J Immunol Methods 113(1) 113-122. 

Phillips, T. M. Dickens, B. F. Affinity and immunoaffinity purification techniques, The Biotechniques Series on Molecular Laboratory Methods -, Eaton Natick, MA, 2000. 

Immunoaffinity Purification and Quantification of Antibody-Toxin Conjugates... 

CR Harrington, DM O Hara, PE Reynolds. Characterization of a monoclonal antibody and its use in the immunoaffinity purification of penicillin-binding protein 2 of methicillin-resistant Staphylococcus aureus. FEMS Microbiol Lett 53 143-147, 1989. 

The continuing refinement in the selection of reference materials and the production of antibodies to complex protein mixtures has resulted in immunoassay systems of remarkable sensitivity and specificity. In particular, the selection and enrichment of the antibody population by immunoaffinity purification against the reference impurities has afforded an additional level of control over the production and validation of these reagents and served to improve the assay range and sensitivity (6,17). This normalization of the antibody population to a stoichiometric relationship with the reference impurities has suggested the term Antigen Selected Immunoassay (ASIA) for these methods. 

Burks, A.W., Cockrell, G., Connaughton, C., and Helm, R.M. 1994. Epitope specificity and immunoaffinity purification of the major peanut allergen, Ara hi. 7 Allergy Clin Immunol 93 743-750. 

The composition of the lysis solution is dictated by the nature of the proteins under study and the subsequent techniques applied to the sample. One of the major choices to be made is whether or not a detergent is required at this stage. If the membrane and soluble fractions are to be separated the initial cell disruption protocol should not include a detergent, as many of the membrane proteins would be solubilized. In this case physical disruption of the cells should be used (e.g., sonication of cells or homogenization of tissues). The choice of lysis conditions is a vital consideration in this work, as proteins need to be solubilized while preservation of posttranslational modifications, inhibition of proteases, maintenance of protein-protein interactions, and, if an immunoaffinity purification step is to be performed, suitability for the antibody to function are essential. For example, SDS is very good at solubilizing membrane proteins but... 

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