title: 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 immunoreactivity. 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.
authors.affiliation: Agouron Institute, La Jolla, CA 92037-4641.
authors.forename: P B
authors.initials: PB
authors.lastname: Billings
authors.forename: A
authors.initials: A
authors.lastname: Martinez
authors.forename: J A
authors.initials: JA
authors.lastname: Haselby
authors.forename: S O
authors.initials: SO
authors.lastname: Hoch
mesh_terms: D001324:Autoantigens / Q000302:isolation & purification*; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D015636:Magnesium Chloride / Q000494:pharmacology; D017830:Octoxynol; D012995:Solubility
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D015636:Magnesium Chloride; D017830:Octoxynol