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A Step-by-Step Guide to the Proximity Ligation Assay (PLA)
- CLYTE research team
- Sep 8
- 5 min read
Ever wondered how scientists can visualize the intricate dance of proteins within a single cell? The Proximity Ligation Assay (PLA) is a powerful technique that allows researchers to do just that. By detecting when two proteins are in close proximity, PLA provides a window into the complex world of protein-protein interactions, modifications, and localization. This guide will walk you through the essential steps of performing a PLA experiment, from sample preparation to data analysis.
The Principle Behind Proximity Ligation Assay: How it Works
At its core, PLA is an antibody-based method. Here's a simplified breakdown of the process:
Primary Antibodies: Two primary antibodies, raised in different species (e.g., rabbit and mouse), recognize the two target proteins of interest.
PLA Probes: Secondary antibodies, called PLA probes, are then added. Each PLA probe is conjugated to a short DNA oligonucleotide (PLUS or MINUS). These probes bind specifically to the primary antibodies.
Ligation: If the two target proteins are close to each other (typically within 40 nanometers), the PLUS and MINUS oligonucleotides on the PLA probes are brought into close proximity. A connector oligonucleotide is added, which hybridizes to the two PLA probes, and a ligase enzyme joins the ends to form a circular DNA molecule.
Amplification: The circular DNA molecule serves as a template for rolling circle amplification (RCA). A DNA polymerase is added, which generates a long, single-stranded DNA product containing hundreds of copies of the original DNA circle.
Detection: The amplified DNA is then detected using fluorescently labeled oligonucleotides that hybridize to the RCA product. The result is a bright fluorescent spot that can be visualized using a microscope. Each spot represents a single protein-protein interaction event.
Step-by-Step Protocol for a Successful PLA Experiment
Here is a general protocol for performing a PLA experiment. Remember to always optimize conditions for your specific antibodies and sample type.
1. Sample Preparation
Cell Culture: Plate cells on coverslips or chamber slides. Aim for a confluency of 50-70% to ensure optimal imaging.
Tissue Sections: For tissue samples, use thin sections (e.g., 40 µm or less) to allow for better antibody penetration.
Fixation: Fix the cells or tissue with a suitable fixative, such as 4% paraformaldehyde (PFA), for 10-20 minutes at room temperature. This step is crucial for preserving cellular structures and protein interactions.
Permeabilization: If your target proteins are intracellular, you'll need to permeabilize the cell membrane. A common method is to use 0.1-0.25% Triton X-100 for 10 minutes at room temperature.
Washing: After fixation and permeabilization, wash the samples thoroughly with a wash buffer, such as PBS (phosphate-buffered saline), to remove any residual reagents.
2. Blocking
Purpose: Blocking is essential to prevent non-specific binding of antibodies, which can lead to high background signal.
Procedure: Incubate your samples with a blocking solution for 1 hour at 37°C in a humidity chamber. Many commercial PLA kits provide an optimized blocking solution.
3. Primary Antibody Incubation
Antibody Selection: Choose primary antibodies that are known to work well for immunofluorescence or immunohistochemistry. You will need two primary antibodies raised in different species.
Incubation: Dilute the primary antibodies in an antibody diluent and incubate the samples overnight at 4°C in a humidity chamber. The optimal antibody concentration should be determined empirically.
4. PLA Probe Incubation
Procedure: After washing off the primary antibodies, incubate the samples with the PLA probes (PLUS and MINUS) for 1 hour at 37°C in a humidity chamber. The PLA probes are secondary antibodies conjugated to oligonucleotides.
5. Ligation
Procedure: Wash the samples and then add the ligation mix, which contains a ligase enzyme and two connector oligonucleotides. Incubate for 30 minutes at 37°C in a humidity chamber. This step will create the circular DNA template.
6. Amplification
Procedure: After another wash step, add the amplification mix, which contains a DNA polymerase and fluorescently labeled oligonucleotides. Incubate for 100 minutes at 37°C in a humidity chamber. This is a light-sensitive step, so protect your samples from light from this point onward.
7. Final Washes and Mounting
Procedure: Perform final washes to remove any unbound reagents. Mount the coverslips onto microscope slides using a mounting medium containing DAPI to stain the cell nuclei.
8. Image Acquisition and Analysis
Microscopy: Visualize the PLA signals using a fluorescence or confocal microscope. The DAPI staining will help you identify the cell nuclei and localize the PLA signals within the cells.
Quantification: The number of PLA signals per cell can be quantified using image analysis software like ImageJ. This data can provide quantitative information about the extent of protein-protein interactions under different experimental conditions.
Frequently Asked Proximity Ligation Assay Questions (FAQ)
How does the proximity ligation assay work?
The Proximity Ligation Assay (PLA) works by detecting two target molecules when they are very close together. First, you use two different primary antibodies, each recognizing one of your target proteins. Then, special secondary antibodies called PLA probes, which have short DNA strands attached, bind to the primary antibodies. If the target proteins are close, these DNA strands are also close. A connector DNA strand is added, and an enzyme called ligase joins everything into a DNA circle. This circle is then amplified thousands of times through rolling circle amplification (RCA). Finally, fluorescent probes bind to this long, amplified DNA, creating a bright spot of light that you can see under a microscope. Each bright spot represents a single interaction event.
What is the distance for proximity ligation assay?
For a signal to be generated in a PLA experiment, the two target proteins must be in very close proximity. The theoretical maximum distance between the two proteins is approximately 40 nanometers (nm). This distance is dictated by the combined size of the primary and secondary antibodies (the PLA probes) that bridge the gap between the two targets. If the proteins are further apart than this, the DNA strands attached to the PLA probes won't be close enough to be ligated into a circle, and no signal will be produced.
Is proximity ligation assay quantitative?
Yes, the proximity ligation assay is considered semi-quantitative. While it provides a digital "yes" or "no" answer for individual interaction events (each fluorescent spot is one event), the overall number of spots can be counted and analyzed. By counting the number of PLA signals per cell or per tissue area, you can compare the relative abundance of protein-protein interactions between different samples or experimental conditions. For example, you could determine if a specific drug treatment increases or decreases the interaction between two proteins. However, it's important to note that it doesn't provide absolute quantification like some other biochemical methods.
What is the PLA method in biology?
In biology, the PLA method is a highly sensitive and specific technique used to visualize and quantify protein-protein interactions, post-translational modifications, and protein localization in situ—meaning directly within fixed cells and tissues. It allows researchers to see where and how often molecules interact within their natural cellular environment. This provides valuable insights into cellular signaling pathways, complex formation, and the functional state of proteins, which is crucial for understanding both normal cellular processes and disease mechanisms.