product line
Duolink®
technique(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
Quality Level
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.
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
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
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.
Other Notes
- 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.
Not included in Detection kit:
Primary antibodies, PLA probes, wash buffers, mounting medium
Legal Information
Cy is a registered trademark of Cytiva
Duolink is a registered trademark of Merck KGaA, Darmstadt, Germany
PLA is a registered trademark of Merck KGaA, Darmstadt, Germany
signalword
Danger
저장 등급
10 - Combustible liquids
wgk
WGK 3
hcodes
Hazard Classifications
Aquatic Chronic 2 - ED ENV 1 - Resp. Sens. 1
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
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
Birgitte B Olsen et al.
BMC molecular biology, 13, 7-7 (2012-03-13)
The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence
Ivan Matic et al.
Molecular cell, 39(4), 641-652 (2010-08-28)
Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry
문서
Support information including tips and tricks, frequently asked questions, and basic troubleshooting.
Learn how Proximity Ligation Assay technology works and how the protein-protein interaction control kit can confirm in situ detection of EGF-induced EGFR-HER2 dimerization.
Things to consider for preparation, setup and execution of the Duolink® assay protocol
프로토콜
This page details the Duolink® In Situ Short Protocol for fluorescence detection
This protocol describes how to perform immunofluorescent detection of proteins in cells and tissue.
관련 콘텐츠
Applications to detect, quantify and visualize protein-protein interactions, post-translational modifications and low expression protein detection using proximity ligation assay
View an automated protocol for Duolink® assays on the AAW™ automated assay workstation and see results comparing manual vs automated runs.
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