Home › Biochemistry › Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions
Biochemistry JoVE (Open Access) Citable · DOI

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

DOI: 10.3791/56457-v
What you'll learn
  • ✓ Design and prepare biotinylated cell-penetrating peptides for intracellular studies
  • ✓ Perform protein-protein interaction assays in living cells using biotin-avidin pull-down
  • ✓ Extract proteins and conduct pull-down analysis to identify interaction partners
  • ✓ Interpret results from intracellular interaction studies in cellular context
Protocol

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Difficulty
advanced
Total time
~4–6 hours per cell line (including incubation and extraction steps)
Model organism
Human glioblastoma stem cells (G166 GSCs)
Biosafety
BSL-1

Steps

1
Prepare cells and biotinylated cell-penetrating peptides

Culture target cells and prepare biotinylated CPP reagents for incubation. Ensure cell viability and peptide characterization prior to treatment.

▶ 00:45
2
Incubate living cells with biotinylated CPP sequences

Treat intact, cultured cells directly with biotinylated cell-penetrating peptides to allow intracellular protein-protein interactions to occur in native cellular environment.

▶ 02:01
3
Extract proteins and perform biotin-avidin pull-down

Lyse treated cells, prepare lysates, and conduct pull-down assay using streptavidin beads to isolate biotinylated peptide-bound protein complexes.

▶ 03:49
4
Analyze and interpret intracellular interaction results

Examine pull-down results from treated cells to identify interaction partners and validate findings specific to intracellular context.

▶ 07:19
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