Published: Vol 5, Iss 10, May 20, 2015 DOI: 10.21769/BioProtoc.1479 Views: 33095
Reviewed by: HongLok LungShannon RuppertAnonymous reviewer(s)
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Abstract
Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell samples. The interaction between two proteins is detected using the corresponding two primary antibodies raised in different species. Species-specific secondary antibodies (PLA probes), each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity (<40 nm), the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. Several-hundredfold replication of the DNA circle can occur after the amplification reaction, and a fluorescent signal is generated by labelled complementary oligonucleotide probes. Therefore, each detected signal is visualized as an individual fluorescent dot, which can be quantified and assigned to a specific subcellular location based on microscopy images. This revolutionary technique enables us to study the protein complex formation with high specificity and sensitivity compared to the other traditional methods, such as co-immunoprecipitation (Co-IP).
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Representative data
Figure 2. Duolink proximity ligation assay for protein interactions between epidermal growth factor receptor (EGFR) and DNA-dependent protein kinase (DNA-PK) in breast cancer cell line MDA-MB-468. The cells were treated with DNA-damaging agent etoposide (20 μM) at different time points, and then Duolink assay between EGFR and IGFBP-3 performed as described (cells without treatment as negative controls). Each red spot represents for a single interaction and DNA was stained with DAPI.
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Acknowledgments
This work was supported by Grant Number DP0984232 to RCB from the Australian Research Council.
References
Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Lin, M. Z., Martin, J. L. and Baxter, R. C. (2015). Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer Cells . Bio-protocol 5(10): e1479. DOI: 10.21769/BioProtoc.1479.
Category
Cancer Biology > General technique > Biochemical assays
Molecular Biology > Protein > Protein-protein interaction
Biochemistry > Protein > Interaction
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