Investigation of ALK2 interactions using a proteomics approach (CaptiVate)
CaptiVate affinity purification coupled with mass spectrometry
Workflow Description
Part I: Bait Construction and Stable Cell Line Generation
- Use of the popular Flp-In system to achieve single-copy, stable expression
- Transgene is inducible thanks to a regulatable Tet-promoter
Part II: Affinity Purification of Bait Protein Complex
Part III: LC-MS/MS Analysis, Data Filtering and Assembly
Analysis of the p53 Complex from Unstimulated HEK293 Cells
- Full-length human p53
- Integration into HEK293
- Two independent rounds of purification and LC-MS/MS
- Background subtraction (180 candidates > 44 candidates)
- Data analysis
CaptiVate™ Platform: Technical Benefits
- Physiological: expression of the bait from a single copy mirrors endogenous expression levels
- Reproducible: all purifications are carried out as biological duplicates (85% reproducibility, TAP tagging: 65% reproducibility)
- Sensitive: Sensitive: only 3×106 cells per purification needed, analysis by LTQ Orbitrap
- Specific: background elimination by proprietary database filtering