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Protein blot assays specific for the discrimination of the centromere autoantigen, CENP‐A, from human cells
Author(s) -
Billings Peter B.,
Martinez Antigona,
Haselby Jeanne A.,
Hoch Sallie O.
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501401145
Subject(s) - centromere , western blot , microbiology and biotechnology , biology , genetics , computational biology , gene , chromosome
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 MgCl 2 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.