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DUO92012 Sigma-Aldrich

Duolink® In Situ Detection Reagents Brightfield

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Properties

Related Categories Duolink PLA Detection Reagents, Duolink PLA Technology, Molecular Biology, Protein Interaction, Proteomics More...
packaging   pkg of Box A (-20°C)
  pkg of Box B (2-8°C)
application(s)   proximity ligation assay: suitable
suitability   suitable for brightfield

Description

Components

This product is comprised of the following:
Box A:
• 5x Ligation - Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase
• 1x Ligase (1 unit/μL)
• 5x Amplification - Contains all components needed for Rolling Circle Amplification (RCA) except the Polymerase
• 5x Detection Brightfield - Contains oligonucleotide probes labeled with horseradish peroxidase (HRP) that hybridize to the RCA product
• 1x Polymerase (10 units/μL)

Box B:
• 1x Hydrogen Peroxide - Contains 0.3% hydrogen peroxide
• Substrate Reagents A-D - Contains all substrate components needed for HRP enzymatic reaction
• 1x Nuclear Stain - Contains Mayer’s hematoxylin solution for cell nuclei staining
See datasheet for more information.

Not included in Detection kit:

Primary antibodies, PLA probes, wash buffers, mounting medium

Other Notes

Storage/Stability
Store Box A and all of its 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.
Store Box B and all of its components at 2-8°C.

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 Brightfield Protocol to use this product.

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 HRP for brightfield detection.

Specificity
Brightfield detection reagents are used for samples with significant autofluorescence.

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

Legal Information

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

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


Custom Duolink Services
Protocols & Articles

Articles

Duolink® PLA Applications

With Duolink® PLA, you can detect, quantify, and visualize protein-protein interactions, post-translational modifications, and low expression protein detection with an easy-to-execute immunodetection...
Keywords: Acylations, Amplification, Biological processes, Buffers, DNA replication, Electrophoresis, Flow cytometry, Gel electrophoresis, Gene expression, Glycosylations, High performance liquid chromatography, Immunohistochemistry, Mass spectrometry, Metabolism, Microscopy, Phosphorylations, Post translational modifications, Sulfations, Transcription, Transduction, Ubiquitinations

Duolink® References

Duolink proximity ligation assay (PLA) has been used in countless studies in a variety of fields. Browse the following tables to view highlights from the many applications of this technology.
Keywords: Apoptosis, Cancer, Cell biology, Coagulation, DNA replication, Degradations, Epigenetics, Flow cytometry, Gene expression, Growth factors, Immunology, Methylations, Neuroscience, Oxidations, PAGE, Peptide synthesis, Phosphorylations, Polymorphisms, Post translational modifications, Transcription

Protocols

Duolink® PLA Brightfield Protocol

This protocol describes the use of Duolink® PLA reagents for the brightfield detection, visualization, and quantification of individual proteins, protein modifications, and protein interactions in ti...
Keywords: Amplification, Cancer, Dehydration reaction, Gene expression, Immunohistochemistry, Peptide synthesis

Duolink® PLA Product Selection Guide

Getting started with Duolink® PLA is simple. The protocols are very similar to that of IF, IHC, and flow cytometry, however Duolink® PLA offers distinct advantages over these and other traditional me...
Keywords: Amplification, Bacterial conjugations, Buffers, Flow cytometry, Fluorescent microscopy, Gene expression, Immunohistochemistry, Microscopy, Peptide synthesis

Duolink® PLA Troubleshooting Guide

The Duolink® PLA technology for protein detection offers a simple and straightforward protocol that has a set of defined steps. This section will delineate important reminders that will ensure your D...
Keywords: Addition reactions, Amplification, Bacterial conjugations, Buffers, Dehydration reaction, Filtration, Flow cytometry, Immunofluorescence, Immunohistochemistry, Peptide synthesis, Purification, Titrations

How to Optimize the Duolink® Proximity Ligation Assay

To successfully execute a Duolink® PLA experiment, there are some necessary considerations to properly prepare for, setup, and execute the assay protocol.
Keywords: Bacterial conjugations, Buffers, Detergents, Flow cytometry, Fluorescent microscopy, Gene expression, Growth factors, Immunofluorescence, Immunohistochemistry, In Situ hybridization, Microscopy, Nucleic acid hybridization, Phosphorylations, Post translational modifications, Sample preparations

Related Content

Duolink® PLA - Protein Detection Technology

Inside every cell, there is a breakthrough waiting to be uncovered. The chance to change the origin of a human disease or discover the tell-tale sign of an irregular protein-protein interaction requi...
Keywords: Amplification, Genetic, Immunofluorescence, Individual protein Immunoprecipitation, Peptide synthesis, Post translational modifications, Protein complex immunoprecipitation, Western blot

Duolink® PLA Custom Services

Duolink® Proximity Ligation Assay (PLA) is a powerful tool that allows in situ detection of endogenous proteins, protein interactions, and protein modifications with high specificity and sensitivity....
Keywords: Flow cytometry, Genetic, Peptide synthesis

Duolink® PLA Resource Center

Understanding protein function in the cell can create solutions to improve human health. For that reason we created the Duolink® PLA Resource Center, to provide insightful educational resources so yo...
Keywords: Flow cytometry, Peptide synthesis

Search Primary Antibodies for use with Duolink® PLA

The use of high quality, primary antibodies is critical to success for running a Duolink® Proximity Ligation Assay. We offer over 300 PLA-validated antibodies and antibody pair kits, in addition to t...
Keywords: Immunohistochemistry, PAGE, Peptide synthesis

Peer-Reviewed Papers
15

References

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