A protocol for immunoaffinity separation of the accumulated ubiquitin-protein conjugates solubilized with sodium dodecyl sulfate.
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.
Shimada
Yohta
Y
Department of Biochemistry, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan.
Fukuda
Takahiro
T
Aoki
Katsuhiko
K
Yukawa
Toyokazu
T
Iwamuro
Shawichi
S
Ohkawa
Kiyoshi
K
Takada
Koji
K
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
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