Methods 1996 Oct 01;102:234-46. doi: 10.1006/meth.1996.0098.

Immunonegative Staining: Epitope Localization on Macromolecules

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Relevant literature relating to immunonegative staining is reviewed and integrated with current research of the author and others. The immunonegative staining procedure has been utilized for the study of epitope localization on immune complexes formed from keyhole limpet hemocyanin type 2 (KLH2) di- and multidecamers, and the 20S and 26S proteasome from Xenopus laevis. The IgG linkage pattern of molecules in small immune complexes is considered to provide the most reliable indication of epitope location. For both KLH2 and the 20S proteasome, using domain-specific monoclonal antibodies and a 32-kDa (p32) subunit-specific polyclonal antibody, respectively, it is shown that epitopes (KLH2, subunit fg domain pair and the proteasome p32) are located on the ends of the two cylindrical molecules. Also, for KLH2 with a monoclonal antibody directed against subunit domain a, it is shown that this domain is located at the center of the KLH2 didecamer. Interpretation of the data for KLH2 indicates that the 10 subunits in each decamer are likely to be oriented in parallel. The epitopes of the 10 fg domain pairs are located on the ends, near the edge of the didecamer cylinder (i.e., at the "collar" region of the didecamer) and the epitope of the 10 a domains is located on the side wall of the didecamer cylinder, where the two decamers meet. In view of the varying shape of the Xenopus 20S proteasome immune complexes, it is tentatively proposed that there may be two p32 subunits located in each of the heptameric alpha rings rather than one, but the possibility of macromolecular heterogeneity with respect to p32 subunit composition cannot be discounted.

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