Hexa-His (His6-Tag) – Catalog #B2026696

Hexa-His (Catalog #B2026696) is a peptide sequence of six consecutive histidine residues (His–His–His–His–His–His) used extensively in protein engineering as a purification tag. Supplied as 1 mg lyophilized powder, this peptide is incorporated into recombinant proteins during expression to enable rapid, single-step purification via immobilized metal affinity chromatography (IMAC). Histidine residues coordinate strongly with divalent metal ions (nickel, cobalt, zinc), allowing tagged proteins to be selectively captured on metal-loaded resins and eluted under mild, non-denaturing conditions. The His6-tag is one of the most widely used affinity tags due to its small size (approximately 0.8 kDa), minimal impact on protein function, and ease of removal if needed.

Scientific Overview

The His6-tag is based on the strong coordination chemistry between histidine imidazole groups and divalent metal ions, particularly nickel (II) and cobalt (II). When a His-tagged protein is loaded onto a metal-loaded chromatography resin (typically Ni-NTA or Ni-IDA), the histidine residues bind to the immobilized metal, while non-tagged proteins flow through. Bound protein is eluted by either increasing imidazole concentration (competitive elution) or decreasing pH to protonate histidine, weakening the metal coordination.

The His6-tag offers several advantages: it is small (0.8 kDa, minimal impact on protein structure and function), widely recognized by antibodies and detection systems, and can be incorporated at either the N- or C-terminus of a protein. Many proteases (e.g., TEV, thrombin) can cleave the tag post-purification if a tag-free protein is desired. IMAC is generally faster and more specific than other affinity tags, making it the standard choice for high-throughput recombinant protein production.

Key applications include:

• Single-step, high-capacity purification of recombinant proteins from crude cell lysates
• Rapid identification and isolation of transiently expressed or weakly expressed proteins
• Protein pull-down and co-immunoprecipitation assays
• Integrated purification and detection workflows

Usage & Handling Guidance

Store lyophilized peptide at –20°C. Before use, dissolve in appropriate buffer (e.g., Tris or phosphate buffer, pH 7–8) to the desired concentration. The His-tag peptide itself is not typically used directly as a purification substrate; instead, it serves as a reference or is incorporated into recombinant proteins via genetic engineering.

• Incorporation into recombinant proteins: The His6 sequence is typically added to expression plasmids via molecular cloning, allowing the target protein to be expressed as a His-fusion during recombinant protein production.
• Purification of His-tagged proteins: Use Ni-NTA, Ni-IDA, Cobalt-IDA, or other metal-loaded resins designed for IMAC.
• Binding: Apply clarified cell lysate to a pre-equilibrated IMAC column; His-tagged protein will bind while non-tagged protein flows through.
• Wash: Wash with binding buffer containing a small amount of imidazole (typically 5–20 mM) to reduce non-specific binding.
• Elution: Elute with higher imidazole concentration (typically 250–500 mM) or lower pH (pH 4.5–5.0).

What You Get

• 1 mg of Hexa-His (His6) peptide, lyophilized
• A reference standard for His-tag detection and identification
• Small, well-characterized peptide tag for recombinant protein engineering
• For research use only (RUO)

Why Researchers Choose It

• Small size (0.8 kDa) minimizes impact on protein structure and function
• Rapid, single-step purification on inexpensive IMAC resins
• Mild elution conditions maintain protein activity and stability
• Wide availability of detection antibodies and compatible reagents
• Can be fused at N- or C-terminus or removed post-purification via protease cleavage

Frequently Asked Questions (FAQ)

• What is the difference between His-tag and His6-tag?
  His6-tag (Hexa-His) specifically refers to six histidine residues in tandem. Longer tags (His8, His10) are also used but are less common. Six histidines provide an optimal balance of binding strength and minimal size impact.
• Can I remove the His-tag after purification?
  Yes. Many proteases (TEV, thrombin, factor Xa, enterokinase) recognize specific cleavage sites engineered between the tag and the target protein, allowing tag removal post-purification.
• Does the His-tag affect protein function?
  Usually not. The His6-tag is small and flexible, but some proteins may be affected, particularly if the tag is positioned near the active site. Testing is recommended.
• What resin should I use for His-tag purification?
  Nickel-NTA or Nickel-IDA resins are the most common; Cobalt-IDA is an alternative with different selectivity and elution characteristics. Choose based on your protein and desired purity.
• What is the typical yield?
  This depends on your expression system, protein stability, and purification conditions. Many users achieve 50–95% recovery of soluble His-tagged protein from clarified lysate.
• Can I get a COA or TDS?
  Yes. Request a quote or contact us and we will provide available lot documentation.

References

• Spriestersbach A, Kubicek J, Schäfer F, Block H, Maertens B. Purification of His-Tagged Proteins. Methods Enzymol. 2015;559:1-15.
• Terpe K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol. 2003;60(5):523-33.
• Loughran ST, Bree RT, Walls D. Poly-Histidine-Tagged Protein Purification Using Immobilized Metal Affinity Chromatography (IMAC). Methods Mol Biol. 2023;2699:193-223.