Protocol for Studying Secretome Interactions
Author Information
Author(s): Rich Joshua A., Gurung Sadeechya, Coates-Park Sasha, Liu Yueqin, Govil Anshika, Stetler-Stevenson William G., Peeney David
Primary Institution: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
Hypothesis
What are the most effective methods for studying the proximal interactomes of secreted factors?
Conclusion
The protocol provides a comprehensive method for studying extracellular proximal interactomes using biotin ligase-based proximity labeling.
Supporting Evidence
- Biotin ligase-based proximity ligation is effective for studying protein interactions.
- The protocol can be adapted for various cell lines and targets.
- Proximity labeling captures weak and transient interactions.
- High expression of fusion proteins is necessary for effective results.
Takeaway
This study shows how to use a special technique to find out how proteins that are secreted from cells interact with each other.
Methodology
The protocol involves steps for cell preparation, sample collection, biotinylated protein enrichment, and post-pull-down processing.
Potential Biases
Potential for background labeling of intracellular targets if not using the conditioned media transfer method.
Limitations
The protocol requires high levels of fusion protein and biotin ligase expression, which can induce toxicity and affect results.
Digital Object Identifier (DOI)
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