Isolation and quantitation of ubiquitin from rat brain

A fast and sensitive method for the isolation and quantitation of cytoplasmic ubiquitin from brain by reversed-phase high-performance liquid chromatography is described. Cytosol from brain tissue was obtained by differential centrifugation and, after perchloric acid treatment, the sample was concentrated and ubiquitin was quantitatively isolated by means of a single chromatographic run. The amino acid composition, molecular weight, and primary structure of the pure protein were identified. The addition of monoiodinated 125I-ubiquitin to the sample as an internal standard indicated high native ubiquitin recovery. Statistical analysis carried out on different preparations and standardization of the chromatographic system indicated both the accuracy and the reproducibility of the method.