A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.
Nat Protoc
; 9(9): 2256-66, 2014 Sep.
Article
in En
| MEDLINE
| ID: mdl-25167057
ABSTRACT
Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Saccharomyces cerevisiae
/
Proteins
/
Chemistry Techniques, Analytical
/
Macromolecular Substances
/
Lipids
Language:
En
Journal:
Nat Protoc
Year:
2014
Document type:
Article
Affiliation country:
Germany