A protocol for immunoaffinity separation of the accumulated ubiquitin–protein conjugates solubilized with sodium dodecyl sulfate

doi:10.1016/j.ab.2008.02.031

Analytical Biochemistry

Volume 377, Issue 1, 1 June 2008, Pages 77-82

https://doi.org/10.1016/j.ab.2008.02.031 Get rights and content

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.

Section snippets

Chemicals and antibodies

Sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate were obtained from Nacalai Tesque (Kyoto, Japan); 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (Chaps) was purchased from Dojindo Laboratories (Kumamoto, Japan); Nonidet P-40 (NP-40) was from Calbiochem (La Jolla, CA); protease inhibitor cocktail (PIC) was purchased from Roche Diagnostics (Indianapolis, IN); cycloamylose was from Ezaki Glico (Osaka, Japan); MG132, phenylmethylsulfonyl fluoride (PMSF), and L-trans

Detergent solubility of TBS-insoluble ubiquitin–protein conjugates in NPC1-deficient mouse

To estimate levels of ubiquitin–protein conjugates, the cerebral extracts were prepared from age-matched wild-type, heterozygous Npc1^+/− and homozygous Npc1^−/− mice by using 2% SDS-containing sample buffer. Smears of ubiquitin–protein conjugates with molecular masses greater than 75 kDa were found in all samples, but the high-molecular weight (HMW) conjugates were more abundant in Npc1^−/− mice than in the wild-type or Npc1^+/− mice (Fig. 1A), suggesting that the treatment with 2% SDS could

Discussion

We report here, to the best of our knowledge, a novel sample preparation method for the immunoprecipitation of ubiquitin–protein conjugates accumulated in the brains of NPC1-deficient mouse and in heat-shocked K562 cells, which are insoluble in Tris-buffered saline but solubilized with 2% SDS. The method is also applicable to the immunoaffinity separation of the 2% SDS-solubilized fraction of membrane raft protein, flotillin-1.

The method comprises two successive steps, the former using an

Acknowledgments

This research was supported by Grants-in-Aid from the Jikei University Research Fund (to K.T.), from the Takeda Science Foundation (to K.T.), and from the Study Group on the Health Effects of Heavy Metals Organized by Ministry of the Environment, Japan (to K.T.). We are grateful to Dr. H. Yokosawa (Hokkaido University) for the generous supply of FK2 antibody and to Ms. T. Hirakawa for her help in animal breeding.

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A protocol for immunoaffinity separation of the accumulated ubiquitin–protein conjugates solubilized with sodium dodecyl sulfate

Abstract

Section snippets

Chemicals and antibodies

Detergent solubility of TBS-insoluble ubiquitin–protein conjugates in NPC1-deficient mouse

Discussion

Acknowledgments

J. Chromatogr. B Anal. Technol. Biomed. Life. Sci.

Int. J. Biochem. Cell. Biol.

Neuron

Am. J. Pathol.

FEBS Lett.

FEBS Lett.

Int. J. Biochem. Cell Biol.

J. Biochem. Biophys. Methods

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biochem. Biophys. Methods

Antibodies: A Laboratory Manual Cold

Isolation of ubiquitin-E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques

Biochem. J.

Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues

Int. J. Cancer