What sequence is a His-tag?
The features
| Features of the His-tag: | |
|---|---|
| Amino Acid Sequence | HHHHHH (4-10) |
| DNA Sequence | 5′-CAT CAC CAT CAC CAT CAC-3′ |
| Size | 840.8 Da (His6) |
| Compatibility to recombinant proteins | Can be added either to the N- or C-Terminus of a protein |
What does His-tag bind to?
The His tag binds to divalent cations immobilized on metal chelation resin, such as nickel resin Ni-NTA (Qiagen GmbH, Germany) or cobalt resin TALON (Clontech, GmbH, Germany). Under our purification conditions (see below) cobalt beads give better results than nickel beads.
What does a His-tag do?
His Tags allow researchers to selectively extract a protein of interest from the thousands of other proteins found in either a cell or cell lysate.
What column is used for His-tag?
The most common ion for his-tag purification of a recombinant protein is Ni2+, though Co2+, Cu2+, and Zn2+ are also used. The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Most other proteins in the lysate will not bind to the resin, or bind only weakly.
Why is His-tag used in protein purification?
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 does his-tagged purification work?
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.
How do you elute His protein tag?
Elution and recovery of captured His-tagged protein from an IMAC column is accomplished by using a high concentration of imidazole (at least 200 mM), low pH (e.g., 0.1 M glycine-HCl, pH 2.5) or an excess of strong chelators (e.g., EDTA). Imidazole is the most common elution agent.
What is His trap column?
HisTrap™ HP is a prepacked, ready-to-use column for the preparative purification of His-tagged recombinant proteins by immobilized metal affinity chromatography (IMAC) using precharged Ni Sepharose™ High Performance.
How does His tag purification work?
How do you purify His tagged proteins?
His-tagged proteins can be purified by a single-step affinity chromatography, namely immobilized metal ion affinity chromatography (IMAC), which is commercially available in different kinds of formats, Ni-NTA matrices being the most widely used.
How do you purify protein with His-tag?
How does His-tag purification work?