Home › Analytical Chem › Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology
Analytical Chem JoVE (Open Access) Citable · DOI

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

DOI: 10.3791/51553-v
What you'll learn
  • ✓ Perform HaloTag protein pulldowns from transfected mammalian cells
  • ✓ Isolate and characterize protein complexes using HaloLink resin
  • ✓ Detect protein interactions via Western blot and mass spectrometry
  • ✓ Visualize fluorescently labeled fusion proteins in live cells
Protocol

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells. Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Difficulty
intermediate
Total time
~4–6 hours per sample (pulldown and imaging combined)
Model organism
Mammalian cells (transfected HEK293 or similar)
Biosafety
BSL-1

Steps

1
Prepare transiently transfected mammalian cells

Transfect mammalian cells with HaloTag fusion protein constructs and culture until sufficient expression is achieved. This generates the protein complexes to be isolated.

▶ 01:23
2
Equilibrate HaloLink resin for binding

Prepare and wash HaloLink resin beads with appropriate buffers to activate the surface and remove storage conditions, enabling efficient capture of HaloTag fusion proteins.

▶ 02:50
3
Bind and wash fusion protein complexes

Incubate cell lysate containing HaloTag fusion proteins with equilibrated HaloLink resin, then perform stringent washes to remove non-specific binding and retain target protein complexes.

▶ 04:01
4
Elute complexes using SDS for analysis

Apply denaturing SDS-containing elution buffer to release captured proteins from the resin for downstream Western blot, mass spectrometry, or gel electrophoresis analysis.

▶ 06:21
5
Image fluorescently labeled fusion proteins in cells

Use fluorescent HaloTag ligands and microscopy to visualize subcellular localization and dynamics of HaloTag fusion proteins in living mammalian cells.

▶ 07:07
6
Analyze protein interactions and localization results

Review Western blot, mass spectrometry, and imaging data to determine interacting partners and subcellular compartmentalization of target HaloTag fusion proteins.

▶ 08:48
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