Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Genetic and biochemical approaches for in vivo and in vitro assessment of protein oligomerization: The ryanodine receptor case study

Stanczyk, Paulina J., Lai, Francis Anthony and Zissimopoulos, Spyros 2016. Genetic and biochemical approaches for in vivo and in vitro assessment of protein oligomerization: The ryanodine receptor case study. Journal of Visualized Experiments (113) , e54271. 10.3791/54271

Full text not available from this repository.

Abstract

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Uncontrolled Keywords: Molecular Biology, Issue 113, chemical cross-linking, co-immnoprecipitation, mammalian cell transfection, oligomerization, ryanodine receptor, self-association, yeast two-hybrid
Publisher: Journal of Visualized Experiments
ISSN: 1940-087X
Last Modified: 27 Mar 2020 02:41
URI: http://orca-mwe.cf.ac.uk/id/eprint/94998

Citation Data

Cited 3 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item Edit Item