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Global Ubiquitination Analysis by SILAC in Mammalian Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1188)

Abstract

Ubiquitination is a versatile and dynamic posttranslational modification in cells, regulating almost all cellular events. With rapid developments of affinity capture reagents and high-resolution mass spectrometry, it is now feasible to globally analyze the ubiquitinated proteome (ubiquitome) using quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC). Here we describe in detail a SILAC protocol to profile the ubiquitome in mammalian cells including protein labeling, antibody-based enrichment, and analysis by mass spectrometry.

Key words

Ubiquitin SILAC Antibody Quantitative proteomics Mass spectrometry 

Notes

Acknowledgements

This work was partially supported by National Institutes of Health grant NS081571 and American Cancer Society grant RSG-09-181, and ALSAC (American Lebanese Syrian Associated Charities).

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Structural Biology, St. Jude Proteomics FacilitySt Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Developmental Neurobiology, St. Jude Proteomics FacilitySt Jude Children’s Research HospitalMemphisUSA

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