In living cells, proteins that function in the same or related pathways work co-operatively by dynamic assembling and disassembling. Especially in the sessile plant lives, biological processes, such as signal transductions and intracellular membrane trafficking, are extensively depended on the interaction of functionally-correlated proteins. Therefore, detection and determination of protein-protein interactions is a critical important approach for the characterization of protein functions.
Pull-down assay is an in vitro method that is widely used for the confirmation or detection of physical interactions between two or more proteins. The principle of the pull-down assay is to utilize affinity ligand to capture interacting proteins.
A standard pull-down assay requires a purified protein with a recombinant affinity tag, which functions as the “bait” and is immobilized to a tag-specific affinity ligand. Then the other protein -- or the “prey” protein, will be incubated with the “bait” protein. Upon physical interactions between these two proteins, the “prey” protein will be captured by the “bait” protein. Following that, the interacting “prey” protein can be eluted using an eluting buffer depending on the affinity ligand. Finally, both the “prey” and “bait” proteins can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then detected by gel staining for western blotting (Figure 1).
Figure 1. Workflow of a standard pull-down assay.
Besides confirmation of protein-protein interactions, the pull-down assay can also be used to characterize interacting conditions. For example, using protein domain truncations to perform pull-down assays can help researchers target the functional regions that are critical for protein interactions. Moreover, the pull-down assay can also be used to explore interactive partners of proteins of interest. Specifically, the “bait” protein is incubated with a mixed protein solution, or cell/tissue extractions. After the washing steps, the bound proteins can be eluted and identified by high-throughput approaches such as liquid chromatography - mass spectrometry (LC - MS).
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