Description
General description
Duolink® In Situ Detection Reagents Far Red contains all the necessary Duolink In situ reagents to perform the amplification and detection of bound PLA® probes. The detection probes contain a fluorophore (lex = 644nm and lem = 669nm), which may be visualized using the same filter as Cy®5. Experiments conducted using Duolink In Situ reagents can detect and visualize protein interactions, protein expression levels and post translational modifications at the single molecule level in fixed cells and tissue samples.
Application
Specificity
FarRed fluorescence detection reagents are often used with Cyanine 5 filter.
Application Note
Two primary antibodies raised in different species are needed. Test your primary antibodies (IgG-class, mono- or polyclonal) in a standard immunofluorescence (IF), immunohistochemistry (IHC) or immunocytochemistry (ICC) assay to determine the optimal fixation, blocking, and titer conditions. Duolink® in situ reagents are suitable for use on fixed cells, cytospin cells, cells grown on slide, formalin-fixed, paraffin embedded (FFPE), or tissue (fresh or frozen). No minimum number of cells is required.
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Duolink®proximity ligation assay(PLA®) allows for endogenous detection of protein interactions, post translational modifications, and protein expression levels at the single molecule level in fixed cells and tissue samples.
Follow the Duolink® In Situ Fluorescence Protocol to use this product. A set of short instructionsis also available.
Visit our Duolink® PLA Resource Center for information on how to run a Duolink® experiment, applications, troubleshooting, and more.
To perform a complete Duolink® PLA in situ experiment you will need two primary antibodies (PLA, IHC, ICC or IF validated) that recognize two target epitopes. Other necessary reagents include a pair of PLA probes from different species (one PLUS and one MINUS), detection reagents, wash buffers, and mounting medium. Note that the primary antibodies must come from the same species as the Duolink® PLA probes. Analysis is carried out using standard immunofluorescence assay equipment.
Features and Benefits
No overexpression or genetic manipulation required
High specificity (fewer false positives)
Single molecule sensitivity due to rolling circle amplification
Relative quantification possible
No special equipment needed
Quicker and simpler than FRET
Increased accuracy compared to co-IP
Publication-ready results
Preparation Note
Storage and Stability: Store the components at –20 °C. The enzymes should be kept cold (–20 °C) at all times, use a freezing block when removing them from the freezer.
Other Notes
5x Ligation - Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase
1x Ligase (1 unit/μL)
1x Polymerase (10 units/μL)
5x Amplification FarRed - Contains all components needed for Rolling Circle Amplification (RCA) except the Polymerase. It also contains oligonucleotide probes labeled with a fluorophore that hybridize to the RCA product.
See datasheet for more information.
Not included in Detection kit:
Primary antibodies, PLA probes, wash buffers, mounting medium
Legal Information
Cy is a registered trademark of Cytiva
Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany
