Home › Microbiology › Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks
Microbiology JoVE (Open Access) Citable · DOI

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

DOI: 10.3791/54870-v
What you'll learn
  • ✓ Generate inducible LAP-tagged stable cell lines for protein studies
  • ✓ Purify LAP-tagged protein complexes using affinity purification
  • ✓ Identify protein-protein interactions via mass spectrometry analysis
  • ✓ Evaluate subcellular localization and protein function in living cells
Protocol

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

Difficulty
advanced
Total time
~4–6 weeks (cell line generation and clonal selection); ~2–4 days (purification and MS analysis per sample)
Model organism
HEK293
Biosafety
BSL-1

Steps

1
Generate inducible stable cell line with LAP-tagged gene

Transfect HEK293 cells with an inducible expression construct encoding the LAP-tagged protein of interest, select stable clones, and validate inducible expression. This establishes the foundational cell line for downstream applications.

▶ 00:44
2
Purify LAP-tagged protein complexes via affinity chromatography

Induce LAP-tagged protein expression, lyse cells under native conditions, and perform affinity purification using the LAP epitope tag to isolate protein complexes while preserving interactions.

▶ 02:06
3
Identify interacting proteins by mass spectrometry

Prepare purified protein complexes for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis to identify and characterize protein-protein interaction networks.

▶ 08:10
4
Evaluate LAP-tagged protein localization and function

Assess subcellular localization via fluorescence microscopy and validate protein function in the inducible stable cell line using biochemical and cell-based assays.

▶ 09:12
💬 Comments coming soon