Purification of His-ubiquitin Proteins from Mammalian Cells
从哺乳动物细胞中纯化带His标签泛素化蛋白
发布: 2012年09月05日第2卷第17期 DOI: 10.21769/BioProtoc.258 浏览次数: 21972
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参见作者原研究论文
The authors used this protocol in:
Jan 2012
Abstract
This protocol is used to purify His-tag ubiquitin conjugated protein. In this particular case, cells were transfected with His-tag ubiquitin and p53 which allowed us to purify using His-tag and reveal the WB using antibodies against p53 to see just the p53-ubiquitinate. The present protocol can be used in general for His-tag proteins expressed in mammalian cells.
Materials and Reagents
- EDTA free protease inhibitors (F. Hoffmann-La Roche, catalog number: 05892988001 )
- Proteasome inhibitor MG-132 (Calbiochem®, catalog number: 474790-5MG )
- Phosphate buffered saline (PBS)
- Tris
- Ni-NTA-Agarose beads (QIAGEN, catalog number: 30210 )
- Urea
- Guanidinium-HCl ( Sigma-Aldrich, catalog number: G4505-25G )
- Imidazole (Sigma-Aldrich, catalog number: 12399 )
- Triton X-100
- Glycerol
- β-mercaptoethanol
- SDS
- Bromophenol blue
- Lysis buffer (see Recipes)
- Wash buffer (see Recipes)
- Elution buffer (see Recipes)
- 4x laemmeli buffer (see Recipes)
Equipment
- Centrifuges
- Sonicator
- Western blotting equipment
- Tissue culture plates
- 1.5 ml eppendorf tube
- 15 ml falcon tube
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Illana, V. O. and Farhaeus, R. (2012). Purification of His-ubiquitin Proteins from Mammalian Cells. Bio-protocol 2(17): e258. DOI: 10.21769/BioProtoc.258.
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分类
生物化学 > 蛋白质 > 分离和纯化
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