proximity ligation assay (PLA)

proximity ligation assay （PLA）(2012-09-07 12:23:18)转载▼分类：science标签：杂谈Proximity Ligation Assay is a technology that extends the capabilities of traditional immunofluorescence and immunohistochemistry to include direct detection of proteins-protein interactions and modifications with unparalleled specificity and sensitivity. Target proteins are detected with single molecule resolution, allowing digital quantification and information about the exact localization of the target.A Revolution in the Detection of ProteinsDuolink™ 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 poss ible 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 differenceThe unique abi ltity 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 PLAUnmodified cellsIn 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 recognitionIn 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 amplificationIn situ PLA technology couples recognition to the possibility to amplify the signal by generating a DNA surrogate of the protein.Localization of the signalIn 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 countingIn 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.Representative image of Proximity LigationAnalysis (PLA) of protein-protein interactionsbetween Caspase 6 (CASP6) and Caspase 3(CASP3). HeLa cells were stained withanti-CASP6 rabbit purified polyclonal antibody1:1200 and anti-CASP3 mouse monoclonalantibody 1:50. Signals were detected byDuolink® 100 Detection Kit 613 (red), andnuclei were counterstained with DAPI (blue).Each red dot represents the detection ofprotein-protein interaction complex. Theimages were analyzed using an optimizedfreeware (BlobFinder) download from TheCentre for Image Analysis at UppsalaUniversity.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 expressionImproving 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.。