How does GST affinity chromatography work?
How does GST-tagged protein purification work? Purification of GST-tagged proteins is based on the affinity of GST to the glutathione ligand coupled to a matrix. The binding of a GST-tagged protein to the ligand is reversible, and the protein can be eluted by adding reduced glutathione to the elution buffer.
What is GST in molecular biology?
In nature, the GST (Glutathione-S-transferase) protein is an enzyme that catalyzes the protective mechanisms of glutathione (GSH). Glutathione is an antioxidant that prevents cell damage by reactive oxygen species. A gst fusion protein is a protein that is tagged with GST protein.
Why does GST bind to the resin?
GST•Bind™ Resin utilizes an 14-atom spacer arm to covalently attach reduced glutathione via a sulfide linkage. The high degree of substitution of glutathione ensures a high binding capacity with yields of glutathione S-transferase (GST) fusion proteins of 5 to 8 mg/ml settled resin.
What does GST protein do?
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 does GST tag bind to?
glutathione
The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure.
What is a 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 is GST assay?
The Glutathione S-Transferase (GST) Assay Kit is intended for the measurement of total GST activity. It can be used to measure GST activity in cell and bacterial lysates, tissue homogenates, and in plasma and erythrocyte lysates.
Is GST a protein?
GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. In addition, GST-tagged fusion proteins can be purified or detected based on the ability of GST (an enzyme) to bind its substrate, glutathione (GSH).
How does GST bind to glutathione?
The 26KDa GST moiety binds with high affinity to glutathione coupled to a Sepharose matrix. This binding is reversible and the protein can be eluted under mild, non-denaturing conditions by the addition of reduced glutathione to the elution buffer.
How do you tag a protein with GST?
To use the GST tag, start by cloning the coding region for the protein of interest into a vector and express the tagged protein in E coli. Using an inducible promoter to control target protein expression can help with proteins that are toxic to bacterial cells.
What are protein tags used for?
Basically, protein tags are peptide sequences that are attached to proteins to facilitate easy detection and purification of expressed proteins. In addition, they can also be used to identify potential binding partners for your protein of interest.