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Updated: Mar 9, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and
Krittalak Chakrabandhu1, Sébastien Huault1, Anne-Odile Hueber2
1Université Côte d'Azur, Institut de Biologie Valrose, CNRS UMR 7277, Inserm UMR 1091, Parc Valrose, Bâtiment des Sciences Naturelles, 06108, Nice, France.
Abstract:
Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).
Insights
Detecting phosphorylated tyrosines in CD95 death domains is key for understanding cell fate. New methods allow specific detection of these phosphorylated tyrosines using site-specific antibodies and immunoblotting techniques.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- The CD95 receptor plays a crucial role in regulating cell death and survival pathways.
- Phosphorylation of specific tyrosines within the CD95 death domain is a critical regulatory event.
- Understanding these phosphorylation events is essential for deciphering CD95-mediated signaling.
Purpose of the Study:
- To provide detailed protocols for the site-specific detection of phosphorylated tyrosines in the CD95 death domain.
- To enable researchers to distinguish between different phosphorylation states of CD95.
- To facilitate the study of CD95 signaling in cell death and survival.
Main Methods:
- Generation of site-specific monoclonal antibodies against phosphorylated tyrosines of CD95.
- Application of conventional SDS-PAGE for separation by protein size.
- Utilizing phospho-protein affinity and mobility shift SDS-PAGE for separation based on phosphorylation status.
- Detection via immunoblotting.
Main Results:
- Successful site-specific detection of individual phosphorylated death domain tyrosines of CD95.
- Demonstration of distinct detection capabilities using different SDS-PAGE techniques.
- Validation of site-specific antibodies for precise analysis of CD95 phosphorylation.
Conclusions:
- Detailed protocols are provided for reliable site-specific detection of CD95 phosphorylated tyrosines.
- These methods enhance the ability to study CD95 signaling and its role in cell fate decisions.
- The developed techniques offer valuable tools for immunological and cell biology research.
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