Duolink™ In Situ Detection Reagents FarRed
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.
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.[1][2][3]
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.
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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• 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
• 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
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.
Cy is a registered trademark of Cytiva
Duolink is a trademark of Sigma-Aldrich Co. LLC
PLA is a registered trademark of Sigma-Aldrich Co. LLC
