title: A protocol for immunoaffinity separation of the accumulated ubiquitin-protein conjugates solubilized with sodium dodecyl sulfate.
abstract: Certain proteins insoluble in aqueous salt solutions are difficult to separate from impurities by immunoaffinity 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 (SDS) 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% SDS. These results indicate that this simple protocol has potential applications for excellent immunoaffinity separation of the less-soluble proteins in diverse cells and tissues.
authors: Department of Biochemistry, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan.
authors: Yohta
authors: Y
authors: Shimada
authors: Takahiro
authors: T
authors: Fukuda
authors: Katsuhiko
authors: K
authors: Aoki
authors: Toyokazu
authors: T
authors: Yukawa
authors: Shawichi
authors: S
authors: Iwamuro
authors: Kiyoshi
authors: K
authors: Ohkawa
authors: Koji
authors: K
authors: Takada
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D018869:Heat-Shock Response; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D047908:Intracellular Signaling Peptides and Proteins; D020014:K562 Cells; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases; D011489:Protein Denaturation; D020673:Protein Renaturation; D011506:Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012995:Solubility; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D003902:Detergents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000077296:Niemann-Pick C1 Protein; C106831:Npc1 protein, mouse; D011506:Proteins; D025801:Ubiquitin; C106354:flotillins; D012967:Sodium Dodecyl Sulfate