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Short answer
A pull-down assay is an in vitro method for testing whether a protein binds partners. A tagged “bait” protein is immobilized on an affinity resin, exposed to a “prey” source such as a cell lysate, and whatever stays bound after washing is eluted and read out on a gel, a Western blot or by mass spectrometry. Its value depends almost entirely on the controls run beside it.
Definition and scope
Thermo Fisher’s instructions for its GST pull-down kit describe the basic pull-down assay as an in vitro technique built on a GST-tagged bait protein (the researcher’s protein of interest) that can be used to identify putative binding partners, the prey. The bait is purified, for example from E. coli or baculovirus-infected insect cells, and immobilized on glutathione affinity resin. It then serves as an affinity support for confirming a suspected protein partner or for finding new ones.
A 2020 review of proteomic approaches in virology (Chappell, White and Damania, Advances in Virus Research) uses the same vocabulary: immunoaffinity purification of protein complexes, also called pull-down assays, rely on a “bait and prey” system in which tagged bait proteins are used to fish for and detect prey proteins.
How a GST pull-down is used in practice
The Thermo Fisher manual lists three sources for the prey: previously purified proteins, cell lysates, and in vitro transcription/translation reactions. Bound proteins are eluted and separated by SDS-PAGE, then visualized by Coomassie, silver or zinc staining, by Western blotting, or by [35S] radioisotopic detection. When the goal is to find new partners, a band can be excised, digested with trypsin and identified by mass spectrometry.
The manual also advises establishing prior experimental evidence that a protein interacts with others before starting, and names yeast two-hybrid, density gradient centrifugation and indirect immunofluorescence as ways to get it. For the two-hybrid approach, see our guide to the yeast two-hybrid assay (Y2H).
Consumables the kit format uses, as listed in the manual: glutathione agarose resin (750 µL settled resin, binding capacity stated as 10 mg GST-tagged protein per mL of settled resin), a lysis buffer, glutathione for elution, TBS, spin columns that retain the resin, and collection tubes. The kit is specified for 25 assays. The wash and bind buffer it describes is 1:1 TBS to Pull-Down Lysis Buffer, giving 75 mM NaCl with 1% of a nonionic detergent, which the manual calls a starting point to be optimized for each protein pair. Elution is with 10 mM reduced glutathione adjusted to pH 8.0 and prepared fresh.
Tags and related methods
GST is one bait tag; the same bait-and-prey logic applies to other affinity tags. Related CASRAI guides cover neighbouring approaches:
- Co-immunoprecipitation (Co-IP), an antibody-based method with its own controls and lysis considerations.
- Tandem affinity purification tagging, a two-step tag-based purification.
- The FLAG tag, a commonly used epitope tag.
- Yeast two-hybrid (Y2H), a cell-based screen suited to establishing prior evidence of an interaction.
Readout by electrophoresis is covered in what is gel electrophoresis.
Controls and false positives checklist
The Thermo Fisher manual states: “Negative controls are absolutely necessary for generating biologically significant results.” It describes two. This table maps each control to the false positive it removes.
| Control | Bait | Prey | What it reveals |
|---|---|---|---|
| Non-treated gel control | Absent | Present | Prey proteins that bind nonspecifically to the immobilized glutathione |
| Immobilized bait control | Present | Absent | Proteins that bind nonspecifically to the GST tag; also confirms the resin captured the bait |
The decision rule in the manual: protein bands that are not present in the control lanes are candidate interacting proteins. Candidates, not confirmed partners.
Matching wash stringency to interaction strength
Appendix A of the manual distinguishes three cases.
| Interaction | What the manual says |
|---|---|
| Strong (low Kd) | Easiest to isolate; can be washed extensively with high-ionic-strength buffer to clear nonspecific proteins |
| Weak (high Kd) | Needs careful evaluation of binding and wash buffer conditions so the interaction stays intact while nonspecific proteins are removed; false positives are more likely, so control design matters |
| Transient | The most challenging; often tied to NTP hydrolysis, so NTP or non-hydrolyzable analogs, cofactors and divalent cations can help trap a complex |
The manual’s troubleshooting table points the same way: high background calls for larger wash volumes or higher ionic strength, while no isolated prey with a weak or transient interaction calls for lower stringency and added cofactors. Appendix B adds that endogenous expression levels vary: an over-abundant prey can mask other bands and an extremely scarce one can fall below detection, so the right amount must be determined empirically.
Common misunderstandings
- “A band means a direct interaction.” The Chappell review notes that affinity purification coupled to mass spectrometry does not indicate whether a protein-protein interaction is direct or indirect, nor the abundance or stoichiometry of the protein.
- “Bands that appear are partners.” Only bands absent from the control lanes count as candidates, and the manual describes using the bait as a way of confirming a previously suspected partner or identifying new ones, not as proof of function.
- “No band means no interaction.” The manual’s troubleshooting table lists weak or transient interactions as a reason no prey is isolated, and calls for lower stringency and added cofactors in that case.
- “A pull-down is the same as Co-IP.” Both are forms of affinity purification, but a pull-down starts from a tagged bait immobilized on resin; see the Co-IP guide linked above for the antibody-based version.
FAQ
What is a pull-down assay used for?
Confirming a suspected protein-protein interaction or identifying new binding partners of a tagged bait protein, using an in vitro bait-and-prey format.
What is the difference between bait and prey?
The bait is the tagged protein of interest immobilized on the affinity resin; the prey is the protein or protein mixture (purified protein, lysate or in vitro translation product) tested for binding to it.
Which controls does a GST pull-down need?
A non-treated gel control (no bait, prey present) and an immobilized bait control (bait present, no prey), per the manufacturer’s instructions cited below.
Does a pull-down band show that two proteins interact directly?
Not by itself. The Chappell review cautions that affinity purification coupled to mass spectrometry does not indicate whether an interaction is direct or indirect, and the Thermo Fisher manual treats bands absent from the control lanes as candidate interacting proteins.
Sources
- Thermo Fisher Scientific, Pierce GST Protein Interaction Pull-Down Kit user guide (MAN0011444): https://documents.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011444_Pierce_GST_Protein_Interact_PullDown_UG.pdf, checked 2026-10-29. Product details above are given as specification, not as a recommendation.
- Chappell DL, White MC, Damania B. Proteomic approaches to investigate gammaherpesvirus biology and associated tumorigenesis. Adv Virus Res 109:201-254: https://pmc.ncbi.nlm.nih.gov/articles/PMC8127003/, checked 2026-10-29.
