Certain proteins insoluble in aqueous salt solutions are difficult to separate from impurities by immuno-affinity techniques, even when the proteins are solubilized with denaturants due to interference of the antigen-antibody reaction. Representative examples of such proteins are the ubiquitin-protein conjugates that accumulate in neuronal tissues of neurodegenerative diseases, the hallmark of such disorders. In this study, we developed a novel sample preparation method comprising two successive steps: Sodium dodecyl sulfate (SIDS) removal from the SDS-containing extracts and renaturation of the denatured proteins. The application of this method was tested on ubiquitin - protein conjugates in the brains of Niemann-Pick type C disease mouse and in heat-shocked K562 erythroleukemia cells. The ubiquitin-protein conjugates in both cases are insoluble in Tris-buffered saline but soluble in 2% SDS. The SDS-solubilized fractions prepared from each of the samples were further pretreated by the method mentioned above, and the ubiquitin-protein conjugates were efficiently immunoprecipitated with the anti-ubiquitin antibody from them. This method was also applied successfully to the immunoprecipitation of flotillin-1, a lipid raft protein, from mouse brain extract prepared with 2% SIDS. These results indicate that this simple protocol has potential applications for excellent immunoaffinity separation of the less-soluble proteins in diverse cells and tissues. (c) 2008 Elsevier Inc. All rights reserved.