What is a 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.

What is a histidine tag used for?

This tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins.

How many kDa is a His tag?

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 does imidazole do in his tag?

Imidazole is utilized as a competitive agent for elution of histidine-tagged proteins. In addition, imidazole can be added in low concentrations in the sample and binding buffer in order to reduce the binding of contaminant proteins, and thus increase the final purity.

How does 6 His tag work?

Some recombinant proteins are engineered to have two hexahistidine tags. 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 big is his tag?

His-tags. Molecular Weight: 0.2–1.6 kDa. 6x-His tag is 0.8 kDa. Tag location: C- or N- terminals, or internal.

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 you install a histidine tag?

Adding polyhistidine tags (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.

Why is histidine attracted to nickel?

A string of histidine residues may be added to the amino or carboxyl terminus of the expressed protein. … This His-tag binds tightly to the immobilized metal ions because the side chain of Histidine, imidazole, has a specific binding affinity to metal ions (in this case, nickel II).

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How do I remove his-tag?

His-tag removal from protein using TEV Protease

  1. Dialyze the protein against 20 mM Tris-HCl, pH 7.5.
  2. Determine the protein concentration.
  3. Combine 15 μg of protein and H2O (if necessary) to make a 45 μl total reaction volume.
  4. Add 5 μl of TEV Protease Reaction Buffer (10X) to make a 50 μl total reaction volume.

What is a recombinant human protein?

What are recombinant proteins? Recombinant proteins are proteins encoded by recombinant DNA that has been cloned in an expression vector that supports expression of the gene and translation of messenger RNA. Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein.

What is nickel NTA?

Ni-NTA Agarose is a nickel-charged affinity resin that can be used to purify recombinant proteins containing a polyhistidine (6xHis) sequence. … Ni-NTA uses the chelating ligand nitrilotriacetic acid (NTA) coupled to a cross-linked 6% agarose resin that is suitable for use in batch and gravity flow applications.

What is imidazole used for?

Imidazole is used to elute tagged proteins bound to nickel ions attached to the surface of beads in the chromatography column. An excess of imidazole is passed through the column, which displaces the His-tag from nickel coordination, freeing the His-tagged proteins.

How does affinity chromatography work?

Affinity chromatography is a separation process used to purify molecules or a group of molecules that are in a biochemical mixture. … The target molecule is then eluted from the ligand by a change made in the buffer conditions so that the protein can be removed from that surface.

What is the underlying principle that allows for the purification of rGFP?

What is the underlying principle that allows for the elution of rGFP from the Ni+2-agarose column? Elution buffer contains imidazole which has a very similar structure as histidine. The agarose column has a greater affinity to the imidazole than the His6 tag.

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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 are purification tags?

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 FPLC system?

Fast protein liquid chromatography (FPLC) is a form of medium-pressure chromatography that uses a pump to control the speed at which the mobile phase passes through the stationary phase. … Since then, many different medium-pressure chromatography systems have been developed.

How do you test for his protein tags?

Detecting His-tagged Fusion Proteins on a Blot Stain the nitrocellulose membrane with 20 ml of ready-to-use InVision™ His-tag In-gel Stain for 20 minutes at room temperature. Rinse the membrane briefly with deionized water. Place the wet or dry membrane on a UV transilluminator equipped with a camera.

What is AviTag?

The AviTag™ is a popular fusion tag due to its powerful and versatile properties. Fused to your protein, the AviTag™ provides a multi-functional system useful for many applications including: Expression. Imaging. Detection.

What are affinity tags?

The affinity tags are unique proteins/peptides that are attached at the N- or C-terminus of the recombinant proteins. These tags help in protein purification. … The epitope tags are generally small peptides with high affinity towards a chromatography resin.

What is Tactin?

The Strep•Tag® II/Strep•Tactin® system combines high specificity with gentle elution conditions to provide highly purified, potentially active, recombinant proteins, or protein complexes, after a single purification step. … The Strep•Tag® system is based on the reliable biotin/streptavidin binding specificity.

What is his-tag name?

A polyhistidine-tag is an amino acid motif in proteins that consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, and by the trademarked name His-tag® (registered by EMD Biosciences).

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How do you remove a histidine tag?

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.

How do you clean histidine tags?

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 does his tag work?

How does histidine-tagged protein purification work? 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.

How does nickel column work?

Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a polyhistidine tag on either terminus. … A recombinant protein with a 6xHis tag has a high affinity for nickel, whereas most other proteins will either bind with low affinity, or not at all.

What functional group of histidine binds to the nickel column?

-nitrilotriacetic acid Histidine (HIS6) Tags The HIS6 binds somewhat specifically to a nickel-nitrilotriacetic acid (NTA) organic functional group that might be bound to an agarose chromatography column, pulling only labeled molecules from the solution phase.

How do you remove imidazole from nickel column?

As far as the resin is concerned, washing the resin with an excess of chromatography buffer after the elution (something simple such as PBS, Hepes-buffered saline etc) is a valid step to remove the vestigial imidazole, prior to washing with a similar volume of mQ water and then storage of the resin with 20% Ethanol.

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