Duolink, In situ PLA

In situ PLA. The PLA probes bind to primary antibodies that are bound to the protein, complex or modification.

PLA™, a proximity ligation assay technology, is capable of detecting single protein events such as protein interactions (e.g. protein dimerization) and modifications (e.g. protein phosphorylation).

The Duolink® reagent series, based on PLA, offers an unprecedented level of sensitivity and specificity for protein detection in fixed cells and tissues. The principle of the technology is based on two unique bi-functional probes called PLA probes. Each PLA probe consists of an antibody attached to a unique synthetic oligonucleotide, which acts as a reporter.
The assay provides exact spatial information on the location of the events and an objective means of quantifying the events.

In principle, with appropriate antibodies, in situ PLA can detect any antigen with proximate epitopes at the single molecule level.

Unique features of in situ PLA

In situ PLA is unique because it requires dual recognition, generates a DNA surrogate of the protein which can then be amplified, and it detects proteins in unmodified cells.

Unmodified cells
In situ PLA can be used on unmodified cells and tissues. The PLA technology works on fresh, frozen and FFPE tissue. Other methods such as BRET, FRET and BiFC act on genetically modified cells with usually overexpressed fusion protein complexes.

Dual recognition
In situ PLA technology requires positive identification of two different epitopes on the same protein or complex, resulting in profoundly enhanced specificity over assays that depend on single binding recognition.

DNA amplification
In situ PLA technology couples recognition to the possibility to amplify the signal by generating a DNA surrogate of the protein.

Localization of the signal
In situ PLA technology generates a localized, discrete signal which is anchored to one of the proximity probes, thereby revealing the exact position of the event.

Digital counting
In situ PLA technology provides an objective means of quantifying and comparing events among different cells, tissues and treatments, which can be automated for screening purposes.
 

In situ PLA shows the exact location of events in intact cells and facilitates quantification and automation of image analysis.

How does in situ PLA work?

Duolink-Discover more about proteins and protein interactions for a step-by-step description of Duolink and in situ PLA technology.

PLA technology compared to other methods

Other methods that enable in-cell visualization of protein complexes include Fluorescent Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET) and Biomolecular Fluorescence Complementation (BiFC). PLA offers significant advantages over BRET, FRET and BiFC. Only in the case of PLA are endogenous, individual protein complexes detected with an amplifiable signal, thereby enabling the detection of single protein events in a quantitative manner.

'Characteristics of in situ PLA compared to other in-cell detection methods' for a comparison with BRET, FRET and BiFC.

'Detection and quantification of HER2 protein using in situ Proximity Ligation Assay' for a comparison with an immunohistochemical approach.