Co-immunoprecipitation for identifying protein-protein interaction on lipid droplets
image: The procedure of lipid droplet protein co-immunoprecipitation. The experimental process includes ten steps that are indicated in the flowchart. These steps can be summarized into seven key components as illustrated in the figure. They are LD isolation, treatment LD with Triton, centrifugal separation of the lipid layer and protein solution, removal of the lipid layer and collection of LD protein-containing solution, binding of LD proteins to antibody beads, washing of non-specific proteins, and elution of the bait-prey complex. The co-IP samples obtained from the experiment are used for SDS-PAGE analysis and MS-based proteomics
Credit: Xiaochuan Fu, Shuyan Zhang and Pingsheng Liu
The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. They have developed an effective method to uncover protein–protein interactions and protein complexes specifically on LDs. In this method, they conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, they begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.
The work entitled “Co-immunoprecipitation for identifying protein-protein interaction on lipid droplets”was published on Biophysics Reports (published on Apr. 2024).