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