How does polyhistidine tag work?

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.

What are histidine tags used for?

The histidine tag The DNA sequence specifying a string of six to nine histidine residues is frequently used in vectors for production of recombinant proteins. The result is expression of a recombinant protein with a 6xHis or poly-His-tag fused to its N- or C-terminus.

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

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 purpose of 6xHis tag?

The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purification tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin for purification.

How does His-tag 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 does Ni NTA column 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.

How does Ni-NTA column work?

What is His tag sequence?

Features of the His-tag: Amino Acid Sequence. HHHHHH (4-10) DNA Sequence. 5′-CAT CAC CAT CAC CAT CAC-3′

Where are his tags added?

(A) The His-tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The His-tag is added using primers containing the tag, after a PCR reaction the tag gets fused to the N-terminus of the gene.

What is NTA in Ni-NTA?

Nitriloacetic acid (NTA) is a chelating agent that forms coordination compounds with metal ions. Nickel (Ni2+) is most commonly used. The resulting complex interacts with histidines due to the intrinsic ability of the imidazole groups to chelate nickel(II) nitriloacetate.

What is tag in protein purification?

Protein tags are most frequently used to purify proteins for which no protein-specific antibody exists. Such tags include his (polyhistidine), FLAG (DYKDDDDK), GST, and Myc tags, which are fused to proteins of interest using expression vector systems.

What is the V5 epitope?

The V5 tag is derived from a small epitope (Pk) found on the P and V proteins of the paramyxovirus of the simian virus 5 (SV5) family. The V5 tag is generally used with all 14 amino acids (GKPIPNPLLGLDST), but it may also be used with a shorter 9-amino acid (IPNPLLGLD) sequence.

What is His-tag sequence?

How is His-tag added?

What are Ni agarose beads?

Ni-NTA Magnetic Agarose Beads are magnetic particles coated with Ni-NTA Agarose affinity purification matrix. They are used for immobilizing and purifying recombinant proteins carrying a His tag.