Protein blot assays specific for the discrimination of the centromere autoantigen, CENP‐A, from human cells

Abstract

The Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP-A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1 M MgCl₂ to allow for further purification. CENP-A copurifies with the histone proteins, primarily H3 and H4. The two CENP-A-specific protein blot assays separate CENP-A from the histone proteins and enhance CENP-A immuno-reactivity. The first assay is based on the use of acid-urea gels with a Triton X-100 concentration chosen to maximize separation of CENP-A from all the histones. The second assay is based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP-A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP-A antigen.