Identification of interacting proteins using PUP-IT
Protein proximity labeling has been developed to identify protein-protein interactions. Here we report a tagging method termed PUP-IT (pupylation based interaction tagging) where a bacterial PUP ligase is fused to the bait protein and this chimeric protein mediates the covalent modification of prey protein with Pup protein. Pup is a small protein containing 64 amino acids. The N terminus of Pup can be fused to the bacteria-derived biotinylated BCCP domain. Therefore, any protein that modified by Pup can be enriched by streptavidin pull down under denaturing condition, which is often used by other types of sample preparation for mass spectrometry identification.
Here we describe two ways to achieve Pup labeling in cells. One is based on transient transfection and another based on the inducible cell line. After labeling in cells, the Pup labeled target proteins can be enriched by using the same streptavidin pull-down procedure.
Posted 14 Aug, 2018
Identification of interacting proteins using PUP-IT
Posted 14 Aug, 2018
Protein proximity labeling has been developed to identify protein-protein interactions. Here we report a tagging method termed PUP-IT (pupylation based interaction tagging) where a bacterial PUP ligase is fused to the bait protein and this chimeric protein mediates the covalent modification of prey protein with Pup protein. Pup is a small protein containing 64 amino acids. The N terminus of Pup can be fused to the bacteria-derived biotinylated BCCP domain. Therefore, any protein that modified by Pup can be enriched by streptavidin pull down under denaturing condition, which is often used by other types of sample preparation for mass spectrometry identification.
Here we describe two ways to achieve Pup labeling in cells. One is based on transient transfection and another based on the inducible cell line. After labeling in cells, the Pup labeled target proteins can be enriched by using the same streptavidin pull-down procedure.
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