Home › Biochemistry › Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
Biochemistry JoVE (Open Access) Citable · DOI

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

DOI: 10.3791/54271-v
What you'll learn
  • ✓ Perform yeast two-hybrid screening to detect protein self-association in vivo
  • ✓ Execute co-immunoprecipitation and chemical cross-linking to validate oligomerization in mammalian cells
  • ✓ Determine homo-oligomer stoichiometry using complementary biochemical methods
  • ✓ Analyze ryanodine receptor homo-tetramer formation and calcium release function
Protocol

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Difficulty
advanced
Total time
~5–7 days (including yeast growth, mammalian cell culture, and biochemical assays)
Model organism
Saccharomyces cerevisiae (yeast), HEK293 (mammalian cell line)
Biosafety
BSL-1

Steps

1
Transform yeast with ryanodine receptor constructs

Prepare competent yeast cells and introduce Y2H expression vectors containing ryanodine receptor domains to enable detection of protein–protein interactions.

▶ 01:10
2
Perform colony-lift filter paper β-galactosidase assay

Transfer yeast colonies to filter paper and incubate with X-gal substrate to identify positive interactions indicated by blue color development.

▶ 03:22
3
Express ryanodine receptor in mammalian cell line

Culture HEK293 cells and transfect with ryanodine receptor expression constructs to generate sufficient protein for biochemical analysis.

▶ 04:29
4
Perform co-immunoprecipitation to detect oligomers

Lyse transfected cells, use antibodies against ryanodine receptor to pull down protein complexes, and analyze co-precipitated oligomeric forms by Western blot.

▶ 06:56
5
Apply chemical cross-linking to stabilize oligomers

Treat cell lysates or isolated proteins with cross-linking reagents to covalently stabilize transient protein–protein interactions before analysis by gel electrophoresis.

▶ 09:02
6
Analyze ryanodine receptor homo-oligomer stoichiometry

Interpret results from Y2H, co-IP, and cross-linking experiments to determine the homo-tetrameric assembly of the ryanodine receptor and confirm its functional role in calcium release.

▶ 09:46
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