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DUO94004

Sigma-Aldrich

Duolink® flowPLA Detection Kit - FarRed

Duolink® PLA kit for Flow Cytometry with FarRed Detection

product line

Duolink®

technique(s)

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

fluorescence

λex 644 nm; λem 669 nm

suitability

suitable for fluorescence

shipped in

dry ice

storage temp.

−20°C

General description

Duolink® flowPLA Detection Kit contains detection oligonucleotides with a fluorophore (lex = 644 nm/lem = 669 nm).

Specificity

Far Red Fluorescence Detection Reagents
Use appropriate laser for λex 644 nm excitation
Use appropriate filter for λem 669 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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View full Duolink® product list
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 

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 - 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 Sigma-Aldrich Co. LLC

Storage Class Code

10 - Combustible liquids

WGK

WGK 2

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
Louise H Jørgensen et al.
The American journal of pathology, 187(2), 457-474 (2016-12-03)
The cytoskeleton is an integral part of skeletal muscle structure, and reorganization of the cytoskeleton occurs during various modes of remodeling. We previously found that the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC) is up-regulated and
Liza Löf et al.
Scientific reports, 7(1), 623-623 (2017-04-06)
Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1

Articles

How to Optimize Duolink® PLA for Flow Cytometry Detection

Considerations for proper experimental design, preparation and execution of the Duolink® PLA for flow cytometry protocol.

Duolink® PLA Flow Cytometry Kits

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.

Troubleshooting Guide for Duolink® PLA for Flow Cytometry Detection

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.

Protocols

Duolink® PLA Flow Cytometry Protocol

Protocol for use of Duolink® PLA reagents for the detection of individual proteins, protein modifications, and protein-protein interactions within cell populations by flow cytometry.

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