Description
General description
Duolink® flowPLA Detection Kit contains detection oligonucleotides with a fluorophore (lex = 644 nm/lem = 669 nm).
Application
Based on proximity ligation assay (PLA), the Duolink® PLA Technology allows for endogenous detection of protein interactions, post-translational modifications, and protein expression levels at the single molecule level in fixed cells.
Duolink® flowPLA Detection Kits will enable 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, suspended cells, two primary antibodies that specifically recognize your proteins of interest, a pair of PLA probes (one 100RXN PLUS and one 100RXN MINUS), wash buffer, and a Duolink® flowPLA Detection Kit. The flowPLA Kits are available with 5 different fluorophores: Violet, Red, Green, Orange, or FarRed. The flowPLA Kits contain all the necessary reagents to perform the amplification and detection of bound PLA probes by flow cytometry. Analysis is carried out using standard flow cytometry assay equipment. User must provide a fixed cell suspension, primary antibodies, and corresponding PLA Probes.
Follow the Duolink® PLA Flow Cytometry Protocol to use this product.
Visit our Duolink® PLA Flow Cytometry page on how to run a Duolink® flow experiment, applications, troubleshooting, and more.
Application Note
Primary antibodies 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. Flow validated antibodies are recommended.
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Duolink® flowPLA Detection Kit–FarRed has been used in in situ proximity ligation assay:
to detect interaction and complex formation between cellular retinoic acid binding protein 1 (Crabp1) and the components of rapidly accelerated fibrosarcoma (Raf) kinase- MAPK-Erk kinase (Mek) signaling pathway
to study protein interaction in human cultured MOLT-4 cells and HeLa cells
to visualize Beclin-1 protein interaction with 14-3-3t in neurons
to study protein interactions in graft endothelial cells
Biochem/physiol Actions
Far Red Fluorescence Detection Reagents
Use appropriate laser for λex 644 nm excitation
Use appropriate filter for λem 669 nm emission
Features and Benefits
Analyze protein protein interactions with flow cytometry readout
Analyze cell populations with Proximity Ligation Assay
Increased sensitivity due to rolling circle amplification for low abundant targets
No overexpression or genetic manipulation required
Relative quantification possible
Works with any flow cytometer instrumentation
Easy to follow flexible protocol
Publication-ready results
Other Notes
This product is comprised of the following:
5x Detection Solution - FarRed (DUO84004)
5x Ligation Buffer (DUO82009)
5x Amplification Buffer (DUO82050)
Ligase (1U/μL)
Polymerase (10U/μL)
See datasheet for more information.
Legal Information
Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany
