How do you elute GST fusion protein?

How do you elute GST fusion protein?

Elute the fusion protein by resuspending the resin with 1.0 ml glutathione elution buffer per ml bed volume. Incubate 10 min at room temperature. Centrifuge 500 × g for 5 min. Transfer the fusion protein-containing supernatant to a separate tube.

How do I add a GST tag to my protein?

Procedure (general version)

  1. Add to the eluate or column purification 10 cleavage units of thrombin ~10 µl per milligramm of GST fusion protein.
  2. Mix gently and incubate the mixture of room temperature (22°C) for 2-16 hours.
  3. After the digest you can stop the reaction by either adding 1 mM of PMSF or ABESF.

What is the role of GST in protein purification?

The GST tag Protein purification with affinity tags such as glutathione S-transferase (GST), histidine (HIS), and other affinity tags, enables purification of proteins with both known and unknown biochemical properties.

Why is GST tag used?

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 do I remove a GST tag?

Removal of the GST tag is often necessary to be able to perform functional or structural studies of the target protein. Tagged proteins containing a PreScission Protease, thrombin, or Factor Xa recognition site can be cleaved either while bound to Glutathione Sepharose® or in solution after elution.

Why is DTT added to PBS?

DTT is a reducing agent and usage will ensure that the protein is unfolded and soluble, easy to purify. To keep the cysteine side chains in their normal reduced state, a reducing agent such as DTT is included in the purification.

How does GST improve solubility?

Because GST folds rapidly into a stable and highly soluble protein upon translation, inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins than expression without the tag.

How big is his tag?

∼2.5 kDa
His-tags, due to their relatively small size (∼2.5 kDa), are not believed to significantly interfere with the function and structure of a majority of proteins.

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 SUMO and SUMOylation?

Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. This process is called SUMOylation (sometimes written sumoylation).

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