DUO92013

Sigma-Aldrich

Duolink® In Situ Detection Reagents FarRed

NACRES:
NA.32

Quality Level

product line

Duolink®

application(s)

proximity ligation assay: suitable

fluorescence

λex 644 nm; λem 669 nm (Cyanine 5, Zeiss Filter set 50)

suitability

suitable for fluorescence

shipped in

dry ice

storage temp.

−20°C

General description

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.

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.

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.

Let us do the work for you, learn more about our Custom Service Program to accelerate your Duolink® projects

View full Duolink® product list

Features and Benefits

  • 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

Components

  • 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

Preparation Note

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.

Legal Information

Cy is a registered trademark of Cytiva
Duolink is a registered trademark of Sigma-Aldrich Co. LLC
PLA is a registered trademark of Sigma-Aldrich Co. LLC

RIDADR

NONH for all modes of transport

Profiling of protein?protein interactions via single-molecule techniques predicts the dependence of cancers on growth-factor receptors.
Lee H W, et al.
Nature Biomedical Engineering, 2(4), 239-239 (2018)
Oliver Werz et al.
Nature communications, 9(1), 59-59 (2018-01-06)
Proinflammatory eicosanoids (prostaglandins and leukotrienes) and specialized pro-resolving mediators (SPM) are temporally regulated during infections. Here we show that human macrophage phenotypes biosynthesize unique lipid mediator signatures when exposed to pathogenic bacteria. E. coli and S. aureus each stimulate predominantly...
M Aubele et al.
British journal of cancer, 103(5), 663-667 (2010-08-12)
Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression...
Kan V Lu et al.
Cancer cell, 22(1), 21-35 (2012-07-14)
Inhibition of VEGF signaling leads to a proinvasive phenotype in mouse models of glioblastoma multiforme (GBM) and in a subset of GBM patients treated with bevacizumab. Here, we demonstrate that vascular endothelial growth factor (VEGF) directly and negatively regulates tumor...
Michaela Graf et al.
Human reproduction (Oxford, England), 26(1), 59-66 (2010-11-30)
Dysfunction of cellular processes in the testes can lead to infertility, tumourigenesis or other testicular disorders. 14-3-3 proteins are known to play pivotal roles in cellular communication, signal transduction, intracellular trafficking, cell-cycle control, transcription and cytoskeletal structure and have been...
Articles
Protocol for immunofluorescent detection of proteins in cells and tissue
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Find Duolink references based on the type of method used, post translational modification detected, and research focus.
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Things to consider for preparation, setup and execution of the Duolink® assay protocol
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Support information including tips and tricks, frequently asked questions, and basic troubleshooting.
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Protocols
This page details the Duolink® In Situ Short Protocol for fluorescence detection
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Applications to detect, quantify and visualize protein-protein interactions, post-translational modifications and low expression protein detection using proximity ligation assay
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