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#His-tag

3 public questions tagged with this topic.

Protein-Protein Interaction

What is a common tag used for affinity purification?

Affinity purification of multiprotein complexes for interaction studies needs small, specific peptide epitopes that enable immunoprecipitation under mild conditions with minimal interference. FLAG octapeptide DYKDDDDK recognized by high-affinity anti-FLAG M2 antibody permits gentle elution with free FLAG peptide, preserving partner proteins for mass spectrometry. FITC and TRITC are fluorescent isothiocyanate dyes used for labeling, not affinity isolation. EGFP is 27 kDa fluorescent reporter useful for localization and FRET but bulky tag may sterically hinder interactions and is not optimized for resin-based purification.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

In pull-down assays, polyhistidine tag binds to:

Polyhistidine tag, commonly hexahistidine, interacts with immobilized divalent cations through coordination chemistry. Histidine imidazole side chains donate electrons to vacant coordination sites of nickel or cobalt ions chelated by nitrilotriacetic acid resin. This interaction remains stable under physiological pH but reversible by competition with high imidazole or low pH. In pull-down and affinity chromatography, His-tagged bait is immobilized on Ni-NTA beads to capture prey interactors. The small tag minimally perturbs folding and enables IMAC purification under native or denaturing conditions unlike streptavidin or glutathione systems.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Hybridization

Polyhistidine-tagged proteins bind to:

Polyhistidine tags, typically hexahistidine, exploit coordination chemistry between imidazole side chains of histidine and immobilized divalent transition metal ions such as Ni2+ or Co2+ chelated by nitrilotriacetic acid resin. At physiological pH, histidine residues donate electrons to vacant orbitals of metal, forming stable complex that allows selective retention of tagged protein from crude lysate. Elution is achieved with high imidazole concentration or low pH. Avidin, GST, and Protein A are affinity matrices for biotinylated, GST-tagged, and antibody Fc-containing proteins respectively, unrelated to metal affinity chromatography principle.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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