Abstract
Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
Keywords: Ubiquitination, Mammalian Cells, in vivo
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Knockdown of TUSC4 expression enhanced BRCA1 polyubiquitination, leading to BRCA1 protein degradation NPRL2/TUSC4 knockdown increased ubiquitination level of BRCA1 compared with control cells (after MG132 enrichment for ubiquitination) (Peng et al., 2015).
Notes
Recipes
Acknowledgments
This work was supported by The Department of Defense (DOD) Breast Cancer Research Program (BCRP) Era of Hope Scholar Award (W81XWH-10-1-0558; S.-Y. Lin).
References
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hello, Xiao Xiao,The full length of 76 AA coded for ubiquitin was used for the plasmid construction, hope this information helps. good luck for your experiment.Yang