Description
Application
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.
It is recommended that Duolink® compensation beads should be used when performing a multiplexed experiment (DUO96001) to save on time and reagent cost, rather than using precious sample. This product can be used to determine flow cytometry instrumentation collection parameters, inform experimental design, and as a control for compensation data for multiplexed FlowPLA experiments.
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 flow cytometry equipment. Duolink® Multicolor flow PLA Reagent Pack Kits used with Duolink® PLA Multicolor Probemaker Kits will enable Multiplexing of up to 4 protein events with sensitive detection of proteins, protein-protein interactions, and protein modifications within cell populations by flow cytometry.
To perform a Duolink® flowPLA experiment, you will need fixed and permeabilized suspended cells, and at least two primary antibodies that specifically recognize your proteins of interest. Analysis is carried out using standard flow cytometry assay equipment. User must provide a fixed cell suspension and primary antibodies. 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). Flow validated antibodies are recommended.
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Features and Benefits
Save time and reagent cost by utilizing beads for compensation rather than precious sample and multiplex reagents
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
Other Notes
This product is comprised of the following:
6.0 – 8.0μm polystyrene beads suitable for use in flow cytometry assays. The bead slurry should have a concentration of 1.5-3.0 million beads/mL. FarRed beads can be mixed with the negative control compensation (DUO84010), or collected alone.
Legal Information
Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany
