Cambridge Bioscience Ltd [Antibodies - Molecular Biology - Fluorescence

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The Duolink(TM) in situ Proximity Ligation Assay (PLA) from Olink is a system which enables the detection and visualisation of individual proteins, protein modifications and protein complexes in tissue and cell samples prepared for microscopy.
The target is detected using two primary antibodies (provided by the user) raised in different species. The Duolink system provides oligonucleotide labeled secondary antibodies (PLA probes) to each of the primaries which only generate a signal when the two primary antibodies are in close proximity. The signal from each detected pair of primary antibodies is then visualised as an individual fluorescent spot. The number of detection events can be quantified (counted) and assigned to a specific subcellular location based on microscopy images.

Principle of the assay: Typical starting materials are adherent cells, cytospin preparations or tissue sections on a glass slide, fixed, pre-treated and blocked with a blocking reagent according to the requirements of the primary antibodies used.
Fig 1: The samples are incubated with a pair of primary antibodies raised in different species that bind to the protein(s) to be detected.
Fig 2: Secondary antibodies conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS) are added to the reaction and incubated.
Fig 3: The Hybridization solution, consisting of two oligonucleotides (red bands), is added and allowed to hybridize to the two PLA probes
Fig 4: The Ligation solution is added together with Ligase (yellow), joining the two hybridized oligonucleotides to a closed circle when the PLA probes are in close proximity.
Fig 5: The Amplification solution, consisting of nucleotides (not shown) is added together with Polymerase (yellow). The oligonucleotide arm of one of the PLA probes acts as a primer for a rolling-circle amplification (RCA) reaction using the formed circular oligonucleotide as a template, generating a concatemeric (repeated sequence) product extending from the oligonucleotide arm of the PLA probe.
Fig 6: The Detection solution, consisting of fluorescently labeled oligonucleotides, is added and the fluorescently labeled probes, hybridize to the RCA product. The signal is easily visible as a distinct fluorescent dot and analyzed by fluorescence microscopy.
To perform a complete in situ PLA, you will need a pair of PLA probes (oligonucleotide conjugated secondary antibodies) and a Detection Kit. A full kit is made up of three components: a PLUS secondary a MINUS secondary and a detection kit. All three are available in both 30 and 100 test sizes. The choice of PLA probes depends on the species in which your primary antibodies are raised. Combine one PLA probe MINUS with one PLA probe PLUS against different species for a complete set and then select the appropriate detection kit for the full system. Click here for a full list of 100 size PLUS and MINUS probe secondaries Click here for a full list of 30 size PLUS and MINUS probe secondaries Detection Kit Guide The Duolink Detection kit is available with two different fluorophores. Detection kit 613 (ex 598, em 613) detected using the same filters as for e.g. Texas Red. Detection kit 563 (ex 557, em 563) detected using the same filters as for e.g. Cy3. Click here for a full list of detection kits or Click here for the full list of all Olink products For our mini catalogue detailing the system in full and all pricing please click here

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