How might the 6 Histidines called a His-tag help in the purification of a protein?
The histidine tag Expressed His-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions.
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.
What is the purpose of his tag?
One of the most commonly used tags is the polyhistidine tag, also known as His-Tag, which is a string of usually between six and nine histidine residues (see Figure 1 below). This method of tagging is especially useful as it allows for easy purification and detection of the recombinant protein.
What is the primary use of his-tags?
His-tags are often used to identify expressing cells when producing recombinant proteins. For example, using anti-His antibodies, researchers can easily detect a secreted His-tagged protein in cell culture supernatant when selecting suitable clones for expansion.
How do I remove His-Tag from protein?
The tag may be removed by cleavage of a protease site placed between the target protein and the affinity tag, followed by a step to separate the protein and affinity tag.
Why is GST tag used?
A GST-tag is often used to separate and purify proteins that contain the GST-fusion protein. It can be fused to either the N-terminus or C-terminus of a protein.
How do you remove glutathione from GST?
The GST can be removed from the sample by re-chromatography on a glutathione column, and the protein of interest purified to homogeneity by other techniques such as gel filtration or ion exchange.
How is his tag used in protein purification?
His-tagged protein purification requires the His-tag and Ni-NTA interaction, which is based on the selectivity and high affinity of Ni-NTA (nickel nitrilotriacetic acid) resin for proteins containing an affinity tag of e.g. six consecutive Histidine residues.
How are histidine-tagged proteins purified in iMac?
Histidine-tagged proteins are commonly purified using Immobilized Metal Affinity Chromatography (IMAC). IMAC is based on the interaction between amino acid residues and divalent metal ions immobilized on resins. Histidine interacts most strongly, and histidine-tagged proteins have extra high affinity because of the multiple histidine residues.
How is Ni-NTA affinity purification of his tagged proteins performed?
Ni-NTA affinity purification of His-tagged proteins is a bind-wash-elute procedure that can be performed under native or denaturing conditions. Here, protocols for purification of His-tagged proteins under native, as well as under denaturing conditions, are given.
When do you remove his tag from a protein?
The his tag usually does not have to be removed. In some cases where it interferes with the function of the target protein or the target protein needs to be in a native state, recombinant proteases are used to easily remove his tag after a second pass over the resin used to purify the protein of interest.