DUO94003

Sigma-Aldrich

Duolink® flowPLA Detection Kit - Orange

Duolink® PLA kit for Flow Cytometry with Orange Detection

product line

Duolink®

application(s)

flow cytometry: suitable
immunofluorescence: suitable
proximity ligation assay: suitable

fluorescence

λex 554 nm; λem 576 nm

suitability

suitable for fluorescence

shipped in

dry ice

storage temp.

−20°C

Specificity

Orange Fluorescence Detection Reagents
Use appropriate laser for λex 554 nm excitation
Use appropriate filter for λem 576 nm emission

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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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

Components

This product is comprised of the following:
  • 5x Detection Solution - Orange (DUO84003)
  • 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 Sigma-Aldrich Co. LLC

Certificate of Analysis

Certificate of Origin

Sofie Selmer Andersen et al.
Cytokine, 64(1), 54-57 (2013-06-04)
Many cytokine receptors are cell surface proteins that promiscuously combine to form active signalling homo- or heterodimers. Thus, receptor chain dimerization can be viewed as a direct measure of a high probability of intracellular signalling by specific cytokines. Proximity ligation...
Tyler J Burns et al.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 91(2), 180-189 (2017-01-18)
To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating...
Karl-Johan Leuchowius et al.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 75(10), 833-839 (2009-08-04)
Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study...
James D Phelan et al.
Nature, 560(7718), 387-391 (2018-06-22)
B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes...
Valerie Le Sage et al.
Virology, 502, 73-83 (2016-12-26)
Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components...
Articles
Considerations for proper experimental design, preparation and execution of the Duolink® PLA for flow cytometry protocol.
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Traditional flow cytometry has been limited in the ability to detect protein-protein interactions and low abundant proteins events — until now. We have combined Duolink® Proximity Ligation Assay (PLA) with flow cytometry in a convenient kit, making the analysis of protein-protein interactions with flow cytometry readouts a reality.
Read More
General tips and tricks for proper experiment execution, aid in identifying potential problems, and provide solutions to ensure a successful Duolink® PLA experiment for flow cytometry.
Read More
Protocols
Protocol for use of Duolink<sup>®</sup> PLA reagents for the detection of individual proteins, protein modifications, and protein-protein interactions within cell populations by flow cytometry.
Read More

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