α2β1-integrin Clustering and Internalization Protocol
α2β1-整合素的群集和内化实验方案
发布: 2014年04月05日第4卷第7期 DOI: 10.21769/BioProtoc.1088 浏览次数: 9743
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Sep 2013
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Abstract
α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the extracellular epitope) to the α2β1-integrins and then to cluster the α2β1-integrin-bound primary antibodies together by the means of the secondary antibody. Binding is done on ice so that the α2β1-integrins will not internalize before both sets of antibodies are bound. Clustering is known to trigger α2β1-integrin internalization efficiently from the cell surface to the cytoplasm. In this protocol we used antibody-induced clustering of α2β1-integrin in order to quantitate the amount of internalized α2β1-integrins in comparison to cell surface-associated α2β1-integrin.
Keywords: integrin (整合素)
collagen binding (胶原结合)
enterovirus (肠道病毒)
internalization (内化)
clustering (聚类)
Materials and Reagents
- Adherent Cells (e.g. A549, Hela, SAOS) (on small rounded coverslips, grown to subconfluency)
- Ice
- Fraction V (Sigma-Aldrich, catalog number: 85040C )
- Primary antibody (binds to the α2-integrin ectodomain) (e.g. AbD Serotec, catalog number: MCA2025 ) (diluted in medium containing 1 % serum; use: 4-5 µg/ml)
- Two different secondary antibodies that recognize the primary antibody (example: goat anti mouse Alexa- 488 and 555; Life Technologies, catalog numbers: A-11001 and A-21424 ) (diluted in medium containing 1 % serum; use: 1.3 µg/ml)
- Serum (Life Technologies, catalog number: 10270-106 )
- DAPI prolong gold mounting media (Life Technologies, catalog number: P36935 ) or any other mounting media
- Phosphate buffered saline (PBS)
- Culture medium with 0-10% serum (see Recipes)
- 4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P-6148 ) (see Recipes)
Equipment
- Coverslip (Thermo Fisher Scientific, Menzel-Gläzer)
- CO2 incubator
- Confocal fluorescence microscope
Software
- Data analysis by BioImage XD (open-source, http://www.bioimagexd.net/)
- ImageJ
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Marjomäki, V., Karjalainen, M., Upla, P., Siljamäki, E., Rintanen, N. and Turkki, P. (2014). α2β1-integrin Clustering and Internalization Protocol. Bio-protocol 4(7): e1088. DOI: 10.21769/BioProtoc.1088.
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分类
免疫学 > 抗体分析 > 抗体-抗原相互作用
生物化学 > 蛋白质 > 免疫检测 > 免疫染色法
细胞生物学 > 细胞结构 > 细胞表面
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