title: Coated microwell plate-based affinity purification of antigens.
abstract: Antibody-based affinity capture of antigens is widely used in the isolation of antigens from complex mixtures. Antibody and the corresponding antigen are allowed to interact with each other to form immunocomplexes which are then typically captured by protein A or protein G immobilized on beaded support. Antigen capture performed using this method generally requires multiple centrifugation steps and careful pipetting to avoid loss of the bead-bound complexes. This traditional procedure is tedious and not easily reproducible, especially when working with multiple samples. To address these issues we have demonstrated that antigens can be captured with protein A/G, protein G, and high binding-capacity streptavidin 96-well strip-coated plates. The coated plate method of antigen purification is reproducible, within the same experiment and between experiments, due to the uniform binding capacity of the plates and wells. Here we report the use of coated microwell plates for antigen capture and for protein-protein interaction studies with the well-characterized BIR2-SMAC, transferrin receptor/ transferrin, and other systems.
authors.affiliation: Pierce Biotechnology Inc., Rockford, IL 61105, USA. surbhi.desai@piercenet.com
authors.forename: Surbhi
authors.initials: S
authors.lastname: Desai
authors.forename: Boguslawa R
authors.initials: BR
authors.lastname: Dworecki
mesh_terms: D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis / Q000302:isolation & purification*; D051017:Apoptosis Regulatory Proteins; D001666:Binding Sites, Antibody; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism; D002624:Chemistry, Clinical / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010455:Peptides / Q000276:immunology; D011990:Receptors, Transferrin / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism
chemical_list: D000941:Antigens; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; C029152:G-substrate; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D011990:Receptors, Transferrin; D013205:Staphylococcal Protein A; D014168:Transferrin; D019809:Streptavidin