How do I purify GST tagged protein?

How do I purify GST tagged protein?

Glutathione Sepharose resins are often used for purification. 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. Optional removal of the GST tag can be performed on the column or after elution.

How do I purify my GST tag?

GST protein from various sources, both native and recombinantly expressed in Escherichia coli and other host cells, can be purified by affinity chromatography on immobilized glutathione, followed by competitive elution with excess reduced glutathione (Smith et al., 1988).

Why does GST bind to the resin?

3.2 GST tag GST binds to resin immobilized glutathione, and this property is used for affinity purification of GST tagged proteins. After the fusion protein is bound to the resin, it can be eluted under rather mild conditions using free reduced glutathione (10–40 mM) at neutral pH.

Where should the GST be inserted into produce the recombinant protein for affinity purification?

Our method involves the fusion of a GST-tag at the N-terminus and a His-tag at the C-terminus of the protein of interest, for an optimal ratio between quantity and purity of the desired protein. The GST is a long tag (29 kDa), which is highly efficient for purification on glutathione Sepharose beads.

What are protein tags used for?

Protein tags are a useful and convenient tool for improving solubility of recombinant proteins, streamlining protein purification, and allowing an easy way to track proteins during protein expression and purification.

What is his tag protein purification?

His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. His-tagged protein is then eluted with a higher concentration of imidazole.

What is the purpose of using the GST alone?

The use of GST as a fusion tag is desirable because it can act as a chaperone to facilitate protein folding, and frequently the fusion protein can be expressed as a soluble protein rather than in inclusion bodies.

What does a GST tag do?

A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein. The tag is 220 amino acids (roughly 26 kDa) in size, which, compared to tags such as the Myc-tag or the FLAG-tag, is quite large. It can be fused to either the N-terminus or C-terminus of a protein.

How can I make 10mm glutathione reduced?

1. Centrifuge 9000 rpm/500 g for 5 min at RT.
2. Wash beads three times (9000 rpm/500 g for 5 min at RT) with 1X PBS containing 1% Triton X-100.
3. Add 10/20 mM reduced glutathione.
4. Centrifuge at 9000 rpm/500 g for 5 min to sediment the gel, and remove the supernatant.
5. Repeat elution and centrifugation steps twice more.

How do you add protein tags?

To add the His tag to your protein, clone the ORF into a vector that carries the tag. Depending on the promoter used, express the tagged protein in bacterial, mammalian or insect cells. Alternatively, you can use cell-free expression systems for protein expression.

What are two purposes of a Polyhistidine tag added to a protein?

Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. However, this technique is generally considered to be less sensitive, and also restricted by some of the more finicky aspects of this technique.

How do you detect His-tagged proteins?

Check total protein content of the gel by staining the gel with a total protein stain. Load at least 1 picomole of the His-tagged fusion protein for detection. Make sure the His-tag is in frame and the protein is expressed properly. Be sure to wash the gel twice with 20 mM phosphate buffer.

How is the purification of GST tagged proteins done?

Purification of GST-tagged proteins is based on the affinity of GST to the glutathione ligand coupled to a matrix. Glutathione Sepharose resins are often used for purification. 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.

How is the GST tag used in prokaryotic expression?

What is GST Tag? Glutathione-S-transferase (GST), a 26 kDa sequence of 211 amino acids, is another widely used affinity tag that increases solubility of the desired protein. GST tag has affinity for immobilized glutathione and is used for prokaryotic expression more frequently.

How does GST bind to glutathione Sepharose?

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.

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.