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#pull-down assay

3 public questions tagged with this topic.

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

What is the principle of pull-down assay?

Principle of pull-down assay centers on affinity binding between tagged bait protein and immobilized ligand, enabling capture of interacting prey proteins from mixture. Bait is expressed as fusion with GST, His, biotin, or Flag tag and retained on corresponding resin. Incubation with cell lysate allows physiological binding partners to co-precipitate via non-covalent interactions. After washing non-specific proteins, complex is eluted and analyzed by SDS-PAGE, Western blot, or mass spectrometry. Method does not depend on temperature-sensitive dyes, fluorescent resonance energy transfer, or radioactive labeling, but on highly specific tag-ligand affinity chemistry.

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

In pull-down assay, GST binds to:

Pull-down assay is an in vitro affinity capture method to demonstrate protein-protein interactions. Glutathione S-transferase fusion bait protein is expressed recombinantly and immobilized on glutathione conjugated agarose or Sepharose beads. Glutathione, a tripeptide, shows high affinity for GST active site, enabling specific immobilization. When incubated with prey lysate, interacting partners co-precipitate and are eluted for SDS-PAGE or Western blot analysis. Binding to streptavidin, nickel, or biotin represents other tagging systems like biotin-streptavidin or His-tag-Ni-NTA, not relevant to GST-based pull-down methodology.

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.

View Details