Ubiquitination Assay for Mammalian Cells
哺乳动物细胞的泛素化试验
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1880 浏览次数: 23874
评审: HongLok LungYu ChenAndrea Puhar
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参见作者原研究论文
The authors used this protocol in:
Jan 2015
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 (泛素化)
Mammalian Cells (哺乳动物细胞)
in vivo (在体内)
Materials and Reagents
- 10 cm dish
- Microcentrifuge tubes
- Eppendorf tubes
- Agarose beads
- U2OS cells
- HA-Ubiquitin plasmid
- FuGene 6 transfection reagent (Promega Corporation, FuGENE®, catalog number: E2691 )
- Reduced-serum medium (Thermo Fisher Scientific, Gibco®, catalog number: 31985-070 )
- MG132
- 1x PBS
- Trypsin (Thermo Fisher Scientific, Gibco®, catalog number: 25300 )
- IgG control antibody according to the targeted antibody species [e.g., Rabbit IgG (Santa Cruz Biotechnology, catalog number: sc-2027 )]
- Agarose conjugate [e.g., Protein A/G agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )]
- BRCA1 antibody
- RIPA buffer (e.g., Santa Cruz Biotechnology, catalog number: sc-24948 )
- Anti-ubiquitin or anti-HA antibody
- Tris-HCl
- SDS
- Bromophenol blue
- β-mercaptoethanol
- Glycerol
- Loading buffer (see Recipes)
Equipment
- Tube rotator located in cold room 4 °C
- Centrifuges (Eppendorf, model: 5424/5424 R )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Peng, Y., Wang, E., Peng, G. and Lin, S. (2016). Ubiquitination Assay for Mammalian Cells . Bio-protocol 6(14): e1880. DOI: 10.21769/BioProtoc.1880.
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分类
生物化学 > 蛋白质 > 修饰
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