How Proximity Ligation Assays (PLA) Work

Duolink^® Proximity Ligation Assay (PLA) is a powerful tool that allows in situ detection of endogenous proteins, protein modifications, and protein interactions with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution in unmodified cells and tissues. Typically, two primary antibodies raised in different species are used to detect two unique protein targets (Figure 1A). A pair of oligonucleotide-labeled secondary antibodies (PLA probes) then binds to the primary antibodies (Figure 1B). Next, hybridizing connector oligos join the PLA probes only if they are in close proximity to each other and ligase forms a closed, circle DNA template that is required for rolling-circle amplification (RCA) (Figure 1C). The PLA probe then acts as a primer for a DNA polymerase, which generates concatemeric sequences during RCA (Figure 1D). This allows up to 1000-fold amplified signal that is still tethered to the PLA probe, allowing localization of the signal. Lastly, labeled oligos hybridize to the complementary sequences within the amplicon (Figure 1E), which are then visualized and quantified as discrete spots (PLA signals) by microscopy image analysis (Figure 1F). Duolink^® PLA can be performed on adherent cells, cytospin preparations, and tissue sections on glass slides using immunofluorescence (i.e., green, red, far red, or orange detection) or immunohistochemistry (i.e., peroxidase-catalyzed reaction). In addition, Duolink^® PLA can be performed on blood or suspension cells for detection by flow cytometry and can be used in multi-well plates (e.g., 96- or 384-well) with a high-content screening imager for high-throughput analysis. The high specificity, sensitivity, and versatility of Duolink^® PLA make it an ideal method for pathway analysis, drug screen kinetics, IC₅₀ determination, and target validation.

Figure 1. Schematic of a Duolink^® PLA reaction. (A) Two primary antibodies recognize specific protein(s) of interest in the cell. The yellow and green globules represent two epitopes of a single protein or epitopes on two different proteins that are interacting. (B) Secondary antibodies coupled with oligonucleotides (PLA probes) bind to the primary antibodies. (C) When the PLA probes are in close proximity, connector oligos join the PLA probes and become ligated. (D) The resulting closed, circular DNA template becomes amplified by DNA polymerase. (E) Complementary detection oligos coupled to fluorochromes hybridize to repeating sequences in the amplicons. (F) PLA signals are detected by fluorescent microscopy as discrete spots and provide the intracellular localization of the protein or protein interaction.

Detection of the Dimerization of EGFR and HER2 using Duolink^® Proximity Ligation Assay

The dimerization of epidermal growth factor receptor (EGFR, ErbB1, HER1) and human epidermal receptor 2 (HER2, ErbB2, Neu) is a well-known protein-protein interaction (PPI). EGFR and HER2 are members of the ErbB family of receptor tyrosine kinases that play key roles in regulating cell division and death. Mutations that affect the expression or activity of either of these proteins are associated with the development of a variety of cancers. Upon binding of ligand (e.g., EGF) to the extracellular domain of EGFR, the receptor becomes activated and autophosphorylates its cytoplasmic tail. This induces the formation of homo- (EGFR-EGFR) and hetero- (e.g., EGFR-HER2) dimers with other members of the ErbB family (Figure 2). Dimerization and subsequent phosphorylation lead to the recruitment of adaptor proteins, activation of intracellular signaling cascades, and, ultimately, changes in gene transcription that can cause cancer cell proliferation, angiogenesis, invasion, metastasis, and reduced apoptosis.

Figure 2. Schematic of EGFR-HER2 dimerization. Ligands, such as epidermal growth factor (EGF) bind to the extracellular domain of EGFR, resulting in the activation and autophosphorylation. This, in turn, results in EGFR homodimerization (not depicted) or heterodimerization with other ErbB family members, such as HER2. Dimerization and subsequent phosphorylation lead to the activation of downstream signaling pathways.

Image Source: Lancet Oncol. 16(15):e543-e554

Duolink^® PLA Control Kit – PPI Confirms Your Experiment is Working

SK-OV3 human ovarian cancer cells express moderate-to-high levels of EGFR and HER2. To confirm in situ detection of EGF-induced EGFR-HER2 dimerization, Duolink^® PLA Control Kit – PPI was used. Each kit contains two 8-well chamber slides with EGF-treated, pre-fixed SKOV3 cells and mouse anti-EGFR and rabbit anti-HER2 primary antibodies. The optimal primary antibody concentrations have already been determined and recommendations are provided in the product insert documentation. Following rehydration in 1x PBS, cells were permeabilized with 0.2% Triton X-100 in 1x PBS for 10 minutes at room temperature and then blocked for 1 hour at 37 °C using Duolink^® PLA Blocking Buffer. Cells in duplicate wells were then incubated with both primary antibodies. Technical negative controls included incubation with each primary antibody separately and no primary antibody, performed on duplicate wells (Figure 3). After washing, Duolink^® PLA were performed according to the Duolink^® PLA Fluorescence Protocol. All wells were incubated with Duolink^® anti-rabbit PLUS and anti-mouse MINUS PLA probes, and PLA signals were generated using Duolink^® In Situ Detection Reagent FarRed. Slides were mounted using the Duolink^® In Situ Mounting Media with DAPI and images were acquired using the GE IN Cell Analyzer 2200.

Figure 3. Duolink^® PLA Control Kit – PPI slide layout and sample placement. All wells contain EGF-stimulated, pre-fixed SK-OV3 cells. After permeabilization and blocking, wells 1 and 5 were incubated with both rabbit anti-EGFR and mouse anti-HER2 primary antibodies, wells 2 and 6 were incubated with anti-EGFR antibody alone, wells 3 and 7 were incubated with anti-HER2 antibody alone, and wells 4 and 8 were incubated with antibody diluent only.

Similar PLA results were obtained when the primary antibodies were incubated for 2 hours at 37 °C (Figure 4A-4D) or overnight at 4 °C (Figure 4E-4H), which allows more flexibility for the user. A robust PLA signal was detected in EGF-activated SK-OV3 cells when both anti-EGFR and anti-HER2 primary antibodies were present, indicative of abundant EGFR:HER2 complexes (Figure 4A, 4E). Despite uniform treatment of SK-OV3 cells with EGF, the number of PLA signals/EGFR:HER2 complexes varied among cells. This is likely due to the differential expression of HER2 within the SK-OV3 cell population (data not shown). In contrast, very few PLA signals were detected when only one primary antibody was used or when both primary antibodies were omitted (Figure 4B-D, 4F-H). Of note, too high of primary antibody concentrations can lead to increased number of PLA signals when used separately and/or can lead to coalescence of positive PLA signals which cannot be accurately quantified (data not shown). Therefore, it is important to use the recommended concentrations of primary antibody. Together, these data show that EGFR:HER2 complexes can be detected and quantified in EGF-stimulated SK-OV3 cells using the Duolink^® PLA Control Kit – PPI.

Figure 4. Detection of EGF-induced EGFR:HER2 dimerization using Duolink^® PLA. Slides of EGF-treated, fixed SK-OV3 cells were incubated with mouse anti-EGFR and rabbit anti-HER2 antibodies (A, E), anti-EGFR antibody only (B, F), anti-HER2 antibody only (C, G), or antibody diluent alone (D, H) for 2 hours at 37 °C (A-D) or overnight at 4 °C (E-H). Duolink^® PLA were then performed using anti-rabbit PLUS and anti-mouse MINUS PLA probes. Ten random 20x images per well were acquired using the DAPI (blue nuclei) and Cy5 (red PLA signals) filters. Images are representative from at least 5 independent experiments.

Protein Detection Formats for Duolink^® PLA Products

Duolink^® PLA products are available for fluorescence and brightfield. Fluorescence Duolink^® PLA reagents can currently be used in both microscopy and flow cytometry application formats.

Fluorescence Detection

Fluorescent microscopy is the most commonly used application format for Duolink^® PLA experiments. Much of the protocol is the same when using Fluorescence Duolink^® PLA reagents as traditional immunofluorescence (IF), however, Duolink^® PLA technology exhibits greater specificity and versatility than IF. The added amplification step included in the Duolink^® PLA procedure provides greater sensitivity.

Brightfield Detection

As with traditional immunohistochemistry (IHC), Brightfield Duolink^® PLA is predominantly used on formalin-fixed, paraffin-embedded tissue sections, but is compatible with other sample types. Brightfield Duolink^® PLA detection reagents contain oligonucleotides labeled with horseradish peroxidase (HRP) that, in the presence of an HRP substrate, forms an enzyme:substrate precipitate visible using a light microscope.

Flow Cytometry

Duolink^® Flow PLA Kit is a fast, powerful statistical analysis tool. This kits identifies protein-protein interactions or subcellular localization. Combining Duolink^® PLA with flow cytometry allows collection of quantitative data in a high throughput manner.

How to Get Started with Duolink^® PLA Technology

The Duolink^® PLA Starter Kit contains all the necessary reagents you need to perform a Duolink^® PLA experiment and analyze up to 30 samples. All you have to provide are prepared cells or tissue samples, primary antibodies, and common laboratory equipment.

A Duolink^® PLA Starter Kit includes:

• Two PLA probes: one PLUS and one MINUS (match to the host of the primary antibodies)
• Antibody diluent and blocking buffer
• Detection reagent: choice of red or orange
• Amplification buffer and polymerase enzyme
• Ligation stock and ligase enzyme
• Wash buffers A and B
• Mounting medium with DAPI

In addition to the Duolink^® PLA Protocols for Fluorescence, Brightfield, and Flow Cytometry, be sure to visit our Resource Center for details on how to successfully set up and execute a Duolink^® PLA experiment, including:

• How to Optimize the Duolink® assay
• Duolink^® PLA Product Selection Guide
• Troubleshooting & FAQ
• Duolink^® PLA Instructional Video