The in situ proximity ligation assay (PLA) is a microscopy-based method for detecting two proteins or epitopes that are located within close molecular proximity inside fixed cells or tissue sections. Two primary antibodies bind the targets, and species-specific oligonucleotide-conjugated PLA probes bind the primary antibodies. If the probes are sufficiently close, their oligonucleotides can be ligated into a circular DNA template that is amplified by rolling-circle amplification and visualised as a discrete fluorescent punctum.
Söderberg and colleagues demonstrated that in situ PLA can visualise endogenous protein complexes at single-molecule resolution in cellular and clinical specimens. A PLA signal supports close proximity, often described as roughly within 40 nm depending on antibody/probe geometry, but does not by itself prove direct physical binding.
Important: PLA depends on two well-validated primary antibodies, accessible epitopes after fixation, compatible host species and rigorous controls. A beautiful punctate image is not sufficient evidence without specificity controls.
When to use PLA
- Visualise endogenous protein proximity inside cells or tissues.
- Localise candidate protein complexes to nucleus, membrane or cytoplasmic compartments.
- Compare proximity after ligand stimulation, inhibitor treatment or genetic perturbation.
- Assess proximity involving post-translationally modified proteins when suitable antibodies are available.
- Validate interactions suggested by Co-IP while preserving spatial context.
PLA versus Co-IP
| Feature | PLA | Co-IP |
|---|---|---|
| Readout | Fluorescent puncta in fixed cells/tissue | Biochemical enrichment in lysate |
| Spatial information | Preserved | Lost |
| Direct binding proven? | No | No |
| Key dependency | Two antibodies + probe compatibility | IP antibody + native complex preservation |
| Best use | Where and when proteins are proximal | Whether proteins co-purify in a complex |
Equipment required
- Fluorescence microscope with appropriate filter sets.
- Humidity chamber.
- Cell-culture coverslips/chamber slides or prepared tissue sections.
- Pipettes and low-retention tips.
- Slide jars/racks.
- Image-analysis software capable of puncta segmentation and cell/nucleus counting.
Reagents
- Two target-specific primary antibodies, typically raised in different host species for standard secondary-probe PLA.
- Species-specific oligonucleotide-conjugated PLA PLUS and MINUS probes.
- Fixative.
- Permeabilisation reagent where intracellular targets are measured.
- Blocking buffer.
- Ligation reagent and ligase.
- Rolling-circle amplification reagents and polymerase.
- Fluorescent detection oligonucleotides.
- Nuclear counterstain and antifade mounting medium.
Antibody-pair design
The antibodies determine both biological specificity and geometric feasibility. Two antibodies can be individually excellent in ICC yet fail in PLA if the bound epitopes place the PLA probes too far apart or fixation masks one epitope.
- Confirm each antibody independently by ICC/IF in the same sample preparation.
- Prefer primary antibodies from different host species unless using directly conjugated/custom PLA probes.
- Titrate both antibodies rather than using standard ICC concentrations automatically.
- Confirm that each target is expressed in the same cell population and subcellular compartment.
- Where possible include knockout/knockdown controls for each target.
Detailed in situ PLA protocol
- Prepare sample. Grow cells on coverslips/chamber slides or prepare validated tissue sections.
- Fix. Use fixation compatible with both primary antibodies. Aldehyde fixation is common; optimise duration to preserve morphology without excessive epitope masking.
- Wash. Remove fixative thoroughly.
- Permeabilise if required. Intracellular targets require membrane access. Use the mildest effective detergent.
- Block. Incubate with the validated PLA blocking solution to reduce non-specific antibody/probe binding.
- Add both primary antibodies. Dilute each antibody to its PLA-optimised concentration and incubate under conditions that preserve signal and specificity.
- Wash thoroughly. Residual primary antibody can elevate background.
- Add PLA probes. Apply species-specific oligonucleotide-conjugated PLUS and MINUS secondary probes.
- Incubate. Allow probes to bind primary antibodies under the reagent system's validated conditions.
- Wash. Remove unbound proximity probes.
- Ligate. Add connector oligonucleotides and ligase. Only probes held within effective proximity efficiently form the circular amplification template.
- Wash. Remove unligated material.
- Rolling-circle amplification. Add polymerase and amplification reagents. The local DNA circle produces a concentrated RCA product at the site of proximity.
- Detect. Hybridise fluorescent detection oligonucleotides to the RCA product.
- Counterstain and mount. Apply nuclear stain and antifade medium.
- Image. Acquire all comparable groups with matched microscope settings and sufficient z-resolution for the sample.
- Quantify puncta. Define segmentation and counting criteria before comparing groups.
Essential controls
- No-primary control: measures non-specific probe/ligation/amplification background.
- Single-primary controls: run antibody A alone and antibody B alone; both should show minimal PLA puncta.
- Biological negative control: knockout, knockdown or a perturbation known to disrupt one target or the interaction.
- Positive-control pair: a known proximal protein pair can verify the assay chemistry.
- Independent ICC controls: confirm each primary antibody stains the expected compartment.
Image acquisition and quantification
- Use the same objective, exposure/laser power, gain and z-stack settings across comparative groups.
- Avoid saturated puncta.
- Count puncta per cell, per nucleus or per defined area with a pre-specified segmentation pipeline.
- Analyse biological replicates, not only fields from one coverslip.
- Report how cells were selected and whether analysis was blinded.
- Where treatment alters cell size or number, choose a denominator that remains biologically interpretable.
Interpreting PLA puncta
A punctum indicates that the two detected epitopes were close enough for the PLA probes to support ligation and rolling-circle amplification. It is evidence of proximity in cellular context, not proof that the two proteins make direct physical contact. Colocalisation, crowding within a complex, scaffold-mediated association or compartmental confinement can all generate proximity.
Warnings and common analytical traps
- Do not call puncta “direct binding” without orthogonal evidence.
- Do not skip single-primary controls. They are essential for detecting probe-specific background.
- Do not assume two antibodies from the same species can be used in a standard two-secondary-probe PLA workflow.
- Do not quantify with different imaging thresholds between groups.
- Do not treat more puncta as stronger affinity. Puncta number reflects proximity events, target abundance, accessibility and assay efficiency.
Limitations
- Requires two compatible antibodies and accessible epitopes.
- PLA cannot by itself distinguish direct from indirect proximity.
- Fixation can create or destroy epitope accessibility.
- RCA produces amplified puncta, so signal intensity is not a direct measure of binding affinity.
- Image segmentation can introduce analysis bias if thresholds are selected after viewing group differences.
Troubleshooting
| Problem | Likely cause | What to try |
|---|---|---|
| No puncta in positive sample | One antibody fails after fixation, incompatible species/probe, epitopes too far apart | Validate both antibodies by ICC, verify probe species and test an alternative antibody epitope. |
| High background everywhere | Non-specific antibody/probe binding or insufficient washing | Titrate primaries, strengthen washing and inspect single-primary controls. |
| Signal only in one-primary control | Probe cross-reactivity or primary/secondary mismatch | Review host species and probe specificity; replace problematic antibody/probe. |
| Large variable puncta clusters | Over-amplification, crowded targets or image saturation | Optimise amplification and acquisition; use z-stacks and unsaturated settings. |
| Poor reproducibility | Variable fixation, confluency or image analysis | Standardise sample preparation and lock analysis thresholds before comparison. |
Frequently asked questions
How close do proteins have to be for PLA?
In situ PLA is commonly described as detecting epitopes within roughly 40 nm, although the effective geometry depends on antibody size, epitope position and probe design.
Does PLA prove an interaction?
It supports close molecular proximity. Direct physical interaction requires additional biochemical, biophysical or structural evidence.
Can PLA be used in FFPE tissue?
Yes, provided antigen retrieval, fixation and antibody performance are validated for the tissue and both epitopes remain accessible.
References and journal sources
- Söderberg O et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006;3:995–1000. doi:10.1038/nmeth947.
- Söderberg O et al. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods. 2008. PMID:18620061.
- Investigating Protein Interactions in Single Cells by Proximity Ligation Assay. Methods Mol Biol. PMID:42162554.
Relevant ABMIUM products
PLA needs a pair of primary antibodies that are specific in the same fixed-cell/tissue preparation and compatible with the PLA probe species. ABMIUM stocks IF/ICC- and IHC-compatible antibodies, but individual catalogue products should not be assumed to form a validated PLA pair. Use Find your MATCH with both targets, species and sample type so host species and application evidence can be checked together.
Explore more with ABMIUM
For interaction validation, compare PLA with the Co-IP Protocol, Pull-Down Protocol and ICC Protocol.