What is GST pull-down assay?
GST pull-down assays involve affinity purifications of one or several unknown proteins from a biological sample using a GST-tagged bait protein. The basic principle is that the GST-tagged bait protein binds to its partners, and the resulting complex is captured on beads with immobilized glutathione.
What do pull down assays show?
The pull-down assay is an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners.
What is the function of glutathione S transferase?
Glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that function to protect cellular macromolecules from attack by reactive electrophiles. Specifically, GSTs catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds (Figure 1).
What is the difference between IP and co-IP?
Difference between IP and co-IP is the focus of the experiment. IP is focused on the primary target, which binds the antibody. Whereas, Co-IP targets the secondary targets, which interacts with the primary proteins, instead of antibody.
How will you develop a pull-down assay to identify new protein interaction complexes?
In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate, and after the prescribed washing steps, the complexes are selectively eluted using competitive analytes or low pH or reducing buffers for in-gel or western blot analysis.
What are GST enzymes?
Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants.
What is the difference between reduced glutathione and glutathione?
The key difference between liposomal glutathione and reduced glutathione is that liposomal glutathione is an active form of glutathione that exists encapsulated inside a lipid molecule in order to enhance the absorption, while reduced glutathione is an active form of glutathione that does not undergo encapsulation.
Why does GST bind to glutathione?
Specifically, the function of GSTs in this role is twofold: to bind both the substrate at the enzyme’s hydrophobic H-site and GSH at the adjacent, hydrophilic G-site, which together form the active site of the enzyme; and subsequently to activate the thiol group of GSH, enabling the nucleophilic attack upon the …