What is the purpose of a His tag?
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.
How does Polyhistidine tag work?
Poly-histidine-tag binds to bivalent nickel or cobalt ions chelated by iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) on sepharose resin/agarose, which allows affinity purification of recombinant protein with a poly-his-tag.
How does His tag 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 many Histidines are in His tag?
Typically, the tag is composed of 6–10 consecutive histidines at either terminus of the protein of interest, often separated by a protease-cleavage site. The presence of a His tag enables the use of IMAC for purification.
How do I remove His-tag?
His-tag removal from protein using TEV Protease
- Dialyze the protein against 20 mM Tris-HCl, pH 7.5.
- Determine the protein concentration.
- Combine 15 μg of protein and H2O (if necessary) to make a 45 μl total reaction volume.
- Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.
What is an anti His tag?
Anti His tag Antibodies Like the MBP (maltose binding protein) and GST tag, the His tag is a small epitope tag consisting of between six to nine histidine residues, which is cloned into an expression vector upstream or downstream of a gene of interest.
How do I get rid of his tag?
Why are protein tags useful?
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.
How does NI-NTA chromatography work?
NTA occupies four of six ligand binding sites of the nickel ion, leav- ing two sites free for interaction with the His6-tag. NTA binds metal ions tightly, allowing use of stringent washes. Histidine residues on the tag, connected via a short linker to the C- or N-terminus of the protein, bind to the Ni-ions.