β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
β1 整合素细胞表面免疫共沉淀（选择性免疫共沉淀）
发布: 2013年11月05日第3卷第21期 DOI: 10.21769/BioProtoc.962 浏览次数: 12433
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参见作者原研究论文
The authors used this protocol in:
Jun 2012
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Abstract
Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation.
Keywords: Integrin (整合素)
Immunoprecipitation (免疫共沉淀)
Selective Immunoprecipitation (选择性沉淀)
Materials and Reagents
- Mouse fibroblasts lacking β1 integrin (β1 -/-) or re-expressing wild-type β1 integrin (β1 wt)
Note: These cells are home-made immortalized mouse fibroblasts derived from floxed β1 parental cells. β1 -/- cells are used as negative control. However, the protocol can also be transferred to other mouse cell lines. When cells lacking β1 integrin are not available as negative control one has to include an unrelated antibody (see steps 2 and 4) to monitor for unspecific binding.
- Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX-I (Gibco, catalog number: 31966-021 )
- Fetal Bovine Serum (FBS) (PAA, catalog number: A15-101 )
- PBS (Sigma-Aldrich, catalog number: P4417 )
- 0.5% Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
- Primaquine bisphosphate (Sigma-Aldrich, catalog number: 160393 )
- Protein G sepharose (Protein G sepharose Fast Flow) (Sigma-Aldrich, catalog number: P3296 )
- BCA Protein Assay (Thermo Fisher Scientific, catalog number: 23227 )
- Triton X100
- Tris-HCl
- Na-deoxycholate
- SDS
- Glycerol
- Bromphenol blue
- Mercapthoethanol
- Protease inhibitors (Complete Mini EDTA-free) (Roche, catalog number: 04 693 159 001 )
- Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3) (Sigma-Aldrich, catalog numbers: P5726 and P0044 )
- Talin-1 antibody (1:1,000 for western blotting) (Sigma-Aldrich, catalog number: T3287 )
- SNX17 antibody (1:1,000 for western blotting) (Proteintech, catalog number: 10275-1-AP )
- β1 integrin IP buffer (see Recipes)
- 2x Laemmli sample buffer (see Recipes)
Equipment
- Cell scraper
- 10 cm cell culture dish
- Centrifuge
- 37 °C, 5% CO2 cell culture incubator
- 26-G needle attached to 1-ml syringe
- Heating block (Eppendorf Thermomixer compact or equivalent)
Procedure
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文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Böttcher, R. T. (2013). β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation). Bio-protocol 3(21): e962. DOI: 10.21769/BioProtoc.962.
Download Citation in RIS Format
分类
生物化学 > 蛋白质 > 免疫检测 > 免疫沉淀（IP）
细胞生物学 > 细胞器分离 > 细胞膜
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