Cell Surface Protein Biotinylation and Analysis
细胞表面蛋白质的生物素修饰和分析
发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.857 浏览次数: 34157
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
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Abstract
A great way to specifically isolate and quantify proteins in the cell surface membrane is to take advantage of the biotinylation technique. It consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pull-down. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control).
Sds-page electrophoresis (SDS-PAGE 电泳)
Western blotting (蛋白质印迹法)
Membrane proteins (膜蛋白)
Materials and Reagents
- Human embryonic kidney HEK293 cells (Health Protection Agency Culture Collections, catalog number: 96121229 )
- Biotin reagent: EZ-link Sulfo-NHS-LC-LC-biotin (Thermo-Pierce, catalog number: 21338 )
- Electrophoresis grade Glycine (Sigma-Aldrich, catalog number: G8898 )
- Immobilized/NeutrAvidin Ultralink Resin (Thermo-Pierce, catalog number: 53150 )
- PierceTM BCA Protein Assay Kit (Thermo-Pierce, catalog number: 23227 )
- Polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Sciences, catalog number: RPN303F )
- Nonfat dry milk (AppliChem GmbH, catalog number: A0830 )
- Chemiluminescence substrate: Super signal west femto maximum sensitivity substrate (Thermo Fisher Scientific, catalog number: 34096 )
- X-ray films: Amersham Hyperfilm ECL (GE Healthcare Life Sciences, catalog number: 28-9068-35 )
- Primary antibodies
- Secondary antibodies
- Protease inhibitors Cocktail (Roche Tablets, catalog number: 11 836 170 001 )
- DPBS with calcium and magnesium (DPBS+) (see Recipes)
- Lysis buffer LB1, LB2, LB3 (see Recipes)
- Phosphate-Buffered Saline (PBS) (see Recipes)
- Saline washing solution (SWS) (see Recipes)
- 3x Gel loading buffer (see Recipes)
- SDS-PAGE gel (see Recipes)
- 5x Running buffer (see Recipes)
- 10x Western blot transfer buffer (see Recipes)
Equipment
- 35 mm Dish (Thermo Fisher Scientific, catalog number: 153066 )
- 6-well cell culture plate (BD Biosciences, catalog number: 353046 )
- Cell scrapers (VWR International, catalog number: 734-2603 )
- 96-well Microtest Plate (Sarstedt, catalog number: 82.1581 )
- OrbitTM LS Low Speed Shaker (LABNET, catalog number: S-2030-LS )
- Rotating wheel (Noria R NR50, Ovan, catalog number: 10000-00062 )
- Microcentrifuge R5415 (Eppendorf, catalog number: 022621425 )
- Digital heat block (VWR International, catalog number: 460-3267 )
- Microplate Reader Benchmark plus (Bio-Rad Laboratories, catalog number: 170-6936 )
- Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )
- Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 170-3935 )
- PowerPac HC Power Supply (Bio-Rad Laboratories, catalog number: 164-5052 )
Software
- ImageJ software (available at http://rsb.info.nih.gov/ij ) (National Institute of Health, NIH)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Tarradas, A., Selga, E., Riuró, H., Scornik, F., Brugada, R. and Vergés, M. (2013). Cell Surface Protein Biotinylation and Analysis. Bio-protocol 3(16): e857. DOI: 10.21769/BioProtoc.857.
Download Citation in RIS Format
分类
细胞生物学 > 细胞结构 > 细胞表面
生物化学 > 蛋白质 > 分离和纯化
生物化学 > 蛋白质 > 标记
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