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A Revolution in the Detection of Proteins
Duolink™ enables researchers to study signalling pathways in native cells and tissue in revolutionizing ways. Based on Olink Bioscience’s proprietary in situ Proximity Ligation Assay (in situ PLA), Duolink™ makes it possible to visualize, localize, and quantify individual protein interactions and modifications using standard fluorescence microscopy. This is the first technology that can readily detect and localize protein interactions without the need of tedious genetic engineering to tag and over-express proteins. Thereby, endogenous protein interactions in cells and tissue can be visualized using your regular immuno-staining antibodies combined with the generic Duolink™ kit.
Each spot makes the difference
The unique abiltity of in situ PLA™ to detect protein modifications, interactions, and their location in tissue samples offers exciting new opportunities in biomarker research. By providing the element of dual recognition to in situ analyses, pathology can expand into a new arena of analytes, beyond measuring the abundance of just a single protein.
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
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.
Protein protein interaction immunofluorescence
Representative image of Proximity Ligation Analysis (PLA) of protein-protein interactions between Caspase 6 (CASP6) and Caspase 3 (CASP3). HeLa cells were stained with anti-CASP6 rabbit purified polyclonal antibody 1:1200 and anti-CASP3 mouse monoclonal antibody 1:50. Signals were detected by Duolink® 100 Detection Kit 613 (red), and nuclei were counterstained with DAPI (blue). Each red dot represents the detection of protein-protein interaction complex. The images were analyzed using an optimized freeware (BlobFinder) download from The Centre for Image Analysis at Uppsala University.
Video Protocol
Antibody Pair for Protein Protein Interaction and Protein Phosphorylation
Single Recognition
Single recognition is used to detect one protein in a highly sensitive way. It can also be used to verify the quality of the primary antibodies before performing a Duolink® Dual recognition assay to detect a protein interaction or a protein phosphorylation.
Detection and quantification of protein expression
Improving upon conventional in situ immunofluorescence, Duolink® gives 1000-fold better signal using your existing primary antibody, and much simpler quantification of protein expression per cell, adding objectivity, resolution, and statistical certainty to studies of protein expression. This is achieved by fluorescent labeling of an amplification product, which is generated whenever an antibody binds to a protein molecule. When you want to detect one single protein target in a highly sensitive way, Duolink single recognition is used.
Webinar:
Detect Protein Interactions in Fixed Cells and Tissues by Duolink®
HER2/HER3 interaction in breast cancer tissue detected by Duolink® Detection Kit HRP/NovaRED
Each red dot in the image represents a fluorescent signal from a single SMAD complex formation.
A Revolution in the Detection of Proteins
Single Recognition
Single recognition is used to detect one protein in a highly sensitive way. It can also be used to verify the quality of the primary antibodies before performing a Duolink® Dual recognition assay to detect a protein interaction or a protein phosphorylation.
