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Why does GST bind to the resin?

Why does GST bind to the resin?

GST binds strongly and specifically to chromatography resins coupled with glutathione. Proteins are eluted under mild conditions that preserve protein function, Protein purities higher than 90% can be obtained after a single purification on Glutathione Sepharose.

What is GST resin?

Glutathione Resin (Cat. No. L00206) is an affinity chromatography medium designed for easy, one-step purification of recombinant glutathione S-transferase (GST) fusion proteins and other glutathione binding proteins expressed in E. coli, insect cells and mammalian cells.

What is GST activity?

Glutathione-S-transferases (GSTs) are a group of enzymes that are important in the detoxication of many different xenobiotics in mammals. GST activity was found to be present in plants,1 insects,2 yeast,3 bacteria,4 and in most mammalian tissues, especially in the liver, which plays a key role in detoxification.

What is the GST enzyme?

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).

How do you elute GST?

1. To elute the protein from the GST tag and agarose bead, add 10µl of thrombin (10 units) per mg GST tagged protein. 2. Mix gently and incubate at room temperature for 2-16 hours.

Where is GST found?

Structure. The glutathione binding site, or “G-site”, is located in the thioredoxin-like domain of both cytosolic and mitochondrial GSTs.

What is GST EGFP?

Glutathione S-transferases (GSTs) form a family of eukaryotic and prokaryotic metabolic enzymes known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to various substrates for the purpose of detoxification. The GST-superfolder GFP fusion protein is about 54 KDa in size.

How is a GST-tagged fusion protein purified?

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). Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S-transferase (GST).

Which is the specific substrate for GST transferase?

Glutathione is a tripeptide (Glu-Cys-Gly) that is the specific substrate for glutathione S-transferase (GST). When reduced glutathione is immobilized through its sulfhydryl group to a solid support, such as cross-linked beaded agarose, it can be used to capture pure GST or GST-tagged proteins via the enzyme-substrate binding reaction.

Why are protein denaturants not compatible with GST?

Because binding depends on preserving the essential structure and enzymatic function of GST, protein denaturants are not compatible. After washing an affinity column to remove non-bound sample components, the purified GST-fusion protein can be dissociated and recovered (eluted) from a glutathione column by addition of excess reduced glutathione.

How are GST-tagged proteins used in biological research?

Therefore, this methodology has become a widely used research tool for determining the biological function of uncharacterized proteins. GST is a 211 amino acid protein (26 kDa) whose DNA sequence is frequently integrated into expression vectors for production of recombinant proteins. The result of expression from this vector is a GST-tagged