A pull-down assay is an affinity-purification method used to test or discover protein-protein interactions. A known bait protein is immobilised on a resin or magnetic bead through an affinity tag or other capture chemistry, then incubated with a sample containing potential binding partners (prey). Proteins that remain associated with the bait after washing are eluted and analysed by SDS-PAGE, western blotting or mass spectrometry.
Pull-down assays are conceptually related to co-immunoprecipitation (Co-IP), but the bait is typically captured through an affinity tag rather than an antibody. Common bait formats include GST-tagged proteins on glutathione resin, His-tagged proteins on immobilised metal affinity resin, and biotinylated proteins on streptavidin resin.
Important: there is no single universal binding buffer for all protein pairs. Ionic strength, pH, detergent, cofactors and incubation time can all affect interaction recovery. Published pull-down methods emphasise empirical optimisation for each protein pair, so the workflow below should be treated as a scientifically grounded starting framework rather than a fixed recipe.
TL;DR: protocol at a glance
| Best suited to | Testing direct or candidate bait-prey interactions using an immobilised tagged or labelled bait. |
|---|---|
| Bait formats | GST, His, biotin/desthiobiotin or another validated affinity tag. |
| Readout | Prey enrichment by SDS-PAGE, western blot or LC-MS/MS. |
| Critical variables | Bait folding, tag position, resin chemistry, buffer composition, wash stringency and negative controls. |
| Interpretation warning | A lysate pull-down can still recover indirect complexes; purified bait + purified prey provides stronger evidence of direct binding. |
When to use a pull-down assay
- Confirm a suspected interaction between a known bait and candidate prey protein.
- Screen a cell lysate for proteins that associate with a recombinant bait.
- Compare binding between wild-type and mutant bait proteins.
- Map interaction domains using truncated or mutated bait constructs.
- Test whether cofactors, nucleotides, salts or other conditions alter binding.
- Enrich bait-associated proteins for LC-MS/MS discovery.
Pull-down assay compared with Co-IP
| Feature | Pull-down | Co-IP |
|---|---|---|
| Bait capture | Affinity tag or labelled bait | Antibody against endogenous or tagged bait |
| Bait source | Often recombinant/purified | Usually present in cell/tissue lysate |
| Strength | Controlled bait chemistry and flexible biochemical optimisation | Can preserve native complexes from biological samples |
| Caution | Tag or recombinant folding can affect binding | Antibody can disrupt or bias recovery |
Choosing a bait-capture system
- GST-tagged bait: captured on glutathione resin; widely used for recombinant protein interaction studies.
- His-tagged bait: captured on nickel- or cobalt-chelate resin; useful for compact affinity tags.
- Biotinylated bait: captured on streptavidin resin; extremely high-affinity capture, with desthiobiotin systems available for gentler elution.
- Other affinity tags: FLAG, Strep-tag and related systems can also be adapted where a suitable resin is available.
Equipment required
- Refrigerated centrifuge or microcentrifuge
- Rotator or gentle rocker suitable for 4°C incubation
- Affinity spin columns, magnetic rack or appropriate resin-handling format
- Ice bucket
- Protein concentration assay
- SDS-PAGE/western blot apparatus or access to LC-MS/MS
Reagents
- Purified bait protein or bait-expression preparation
- Affinity resin compatible with the bait tag
- Cell/tissue lysate or purified prey protein
- Binding buffer
- Wash buffer
- Protease inhibitors for lysate-based workflows
- Elution buffer compatible with the affinity tag and downstream analysis
- Positive and negative control bait preparations where available
Design considerations before starting
The biochemical conditions should preserve the interaction without promoting excessive non-specific binding. Start from conditions that are compatible with the biology of the proteins rather than using a highly stringent buffer by default.
- pH: choose a range compatible with both protein stability and the affinity chemistry.
- Salt: around physiological ionic strength is a common starting point, but weak electrostatic interactions can be salt-sensitive.
- Detergent: mild non-ionic detergent may reduce non-specific binding but can disrupt hydrophobic interactions.
- Cofactors: include ATP, Mg²⁺, Ca²⁺, nucleotides, lipids or other cofactors if the interaction is known to require them.
- Reducing agent: use only where compatible with the resin and protein structure.
Prepare the bait protein
- Express and purify the tagged bait protein under conditions that preserve folding and activity.
- Confirm bait purity by SDS-PAGE before immobilisation.
- Where possible, verify that the bait retains biological or binding activity after tagging.
- Prepare an appropriate negative bait control, such as tag alone, unrelated tagged protein or interaction-deficient mutant.
Prepare the prey sample
Cell or tissue lysate
- Harvest cells or tissue and keep material cold.
- Lyse with a mild buffer containing fresh protease inhibitors.
- Clarify by refrigerated centrifugation at a force sufficient to remove insoluble debris without pelleting soluble complexes. Keep the chosen condition consistent across experiments.
- Retain an aliquot as the input sample.
Purified prey protein
Purified prey is useful for confirming a suspected direct interaction because the system contains fewer potential bridging proteins. However, purified-protein interaction results depend strongly on correct folding and may not reproduce the cellular environment.
Pull-down protocol
- Equilibrate the affinity resin. Wash the resin with binding buffer to remove storage solution and establish the correct ionic conditions.
- Immobilise the bait. Add the tagged or labelled bait to the resin and incubate under conditions appropriate for the tag. Do not overload the resin; excessive bait can increase non-specific adsorption and reduce efficient washing.
- Wash away unbound bait. Remove unbound protein with several washes. Retain an aliquot if you need to assess bait-coupling efficiency.
- Add the prey sample. Apply clarified cell lysate, tissue lysate or purified prey to the immobilised bait. Retain an input sample for analysis and keep the bait:prey ratio and sample volume consistent across comparable reactions.
- Incubate to allow binding. Incubate at 4°C with gentle rocking or rotation. One hour is a common minimum starting point in commercial pull-down protocols, but weaker interactions may require longer incubation.
- Collect the resin. Use centrifugation, gravity flow or magnetic separation depending on the resin format.
- Save the flow-through if useful. Flow-through analysis can reveal whether the prey failed to bind or whether the bait/resin was saturated.
- Wash the resin repeatedly. Use several washes with sufficient volume to remove unbound and non-specifically associated proteins. Maintain cold conditions for lysate-derived interactions.
- Optimise wash stringency. If background is high, increase salt concentration modestly, adjust detergent, or increase wash number. If expected prey disappears, reduce stringency.
- Elute bound proteins. Choose a method appropriate to the tag and downstream analysis. Options include competitive ligand, imidazole for His-tag systems, reduced glutathione for GST, low pH, high salt, biotin for selected desthiobiotin systems, or denaturing SDS sample buffer.
- Analyse bait and prey. Run input, negative-control pull-down, bait pull-down and eluate side by side by SDS-PAGE and western blot. For discovery experiments, process the eluate for mass spectrometry.
Essential controls
- Beads/resin-only control: prey sample incubated with resin lacking bait.
- Tag-only control: resin loaded with tag alone or an empty-vector product.
- Input sample: confirms that candidate prey was present before pull-down.
- Irrelevant bait: controls for proteins that bind non-specifically to a recombinant protein of similar size or composition.
- Positive interaction control: useful during assay development.
- Interaction-deficient bait mutant: a particularly strong control when a known binding site can be disrupted selectively.
How to distinguish direct from indirect interactions
Pull-down from a complex cell lysate can still capture proteins that are connected indirectly through another protein or nucleic acid. To strengthen evidence for direct binding, repeat the experiment with purified bait and purified prey under defined conditions. If the interaction depends on DNA, RNA or a cofactor, removing that component can abolish binding even when the proteins associate in cells.
Discovery pull-downs and mass spectrometry
For LC-MS/MS discovery, negative controls and replicates are critical. Compare bait pull-downs with resin-only and tag-only controls. Proteins consistently enriched over controls are stronger candidates than proteins detected only once. Common abundant contaminants should be interpreted cautiously.
Recommendations and warnings
- Always run a tag-only or resin-only control. Sticky proteins can bind the matrix or affinity tag and look like genuine interactors.
- Verify bait loading before testing prey. Inconsistent bait immobilisation can dominate apparent differences in interaction strength.
- Do not assume recombinant bait is native. Folding, oligomerisation and post-translational modifications may differ from the endogenous protein.
- Keep cofactors in the system if biology requires them. Nucleotide-, metal-, lipid- or redox-dependent interactions can disappear in a simplified buffer.
- Use an interaction-deficient mutant when available. It is often more informative than an unrelated bait because it controls for the same protein scaffold and tag.
Limitations
- Recombinant bait may be misfolded or lack native post-translational modifications.
- Affinity tags can sterically interfere with interaction surfaces.
- High-affinity resin capture can favour bait immobilisation but does not guarantee biologically meaningful prey binding.
- Weak interactions can be lost during washing.
- Non-specific proteins can bind directly to resin, tags or highly charged bait proteins.
- A positive lysate pull-down does not necessarily prove direct interaction.
Troubleshooting
| Problem | Likely cause | What to try |
|---|---|---|
| Bait does not bind resin | Tag inaccessible, damaged or incompatible buffer | Confirm tag expression, review resin chemistry and remove incompatible reagents. |
| Expected prey absent | Interaction too weak, bait misfolded or wash conditions too stringent | Verify bait folding, reduce wash stringency and test purified prey or required cofactors. |
| Many background proteins | Non-specific resin/tag binding or overly mild washes | Use tag-only/resin controls, increase salt/detergent modestly and pre-clear lysate. |
| Poor reproducibility | Variable bait loading or protein input | Quantify bait and prey inputs and standardise resin, incubation and wash conditions. |
| Prey in flow-through but not eluate | Prey did not bind or resin capacity was insufficient | Increase immobilised bait within resin capacity or optimise interaction buffer. |
Warnings and common mistakes
- Do not treat a lysate pull-down as proof of direct binding. A third protein, lipid or nucleic acid can bridge bait and prey.
- Do not ignore tag orientation. N- and C-terminal tags can affect folding or mask an interaction surface.
- Do not overload affinity resin. Excess bait can increase aggregation and non-specific adsorption.
- Do not use one wash buffer for every protein pair. Optimise stringency against both background and recovery of the expected prey.
- Do not interpret proteins recovered equally in tag-only or beads-only controls as specific interactors.
Frequently asked questions
Does a pull-down assay prove two proteins bind directly?
Only when the system is sufficiently defined. A pull-down using purified bait and purified prey can support direct binding. A pull-down from cell lysate can recover indirect members of a complex.
Can a pull-down assay estimate binding affinity?
Yes, with purified proteins, a concentration series, controlled bait loading and a saturation analysis. A single endpoint experiment cannot provide a reliable dissociation constant.
Should I use GST, His or an epitope tag?
Choose the system that best preserves bait folding and provides clean controls. GST is relatively large, His tags are compact but metal-affinity resins can have distinct background, and FLAG/Myc/HA systems can simplify immunodetection.
Related ABMIUM protocols
References and journal sources
- Protein-Protein Interactions: Pull-Down Assays. Methods Mol Biol. 2017;1615. PMID:28667618.
- A guide to simple, direct, and quantitative in vitro binding assays. 2019. PMID:31453222.
Relevant ABMIUM products
Use only reagents compatible with the tag and downstream workflow used in your construct.
- FLAG-Tag Mouse mAb — listed for WB, IF and IP workflows.
- C-Myc-Tag Mouse mAb — listed for WB, IF and IP workflows.
- His-Tag Mouse mAb — listed for WB, IF and IP workflows.
- Recombinant FLAG tag Control Protein — useful where a FLAG-tag control is appropriate to the assay design.
- Calibrated Color Prestained Protein Marker (10–250 kDa) — for downstream SDS-PAGE/western blot analysis.
Explore more with ABMIUM
For a tagged interaction assay, use Find your MATCH with the bait, prey and tag system. You can also explore recombinant proteins, search tag antibodies, or continue to the Co-IP Protocol and Western Blot Protocol.