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DUO92012

Sigma-Aldrich

Duolink® In Situ Detection Reagents Brightfield

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Synonym(s):
in situ Proximity Ligation Assay reagent, Protein Protein Interaction Assay reagent

product line

Duolink®

packaging

pkg of Box A (-20°C)
pkg of Box B (2-8°C)

technique(s)

proximity ligation assay: suitable

suitability

suitable for brightfield

storage temp.

−20°C

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DUO92007DUO82049DUO92010
technique(s)

proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

technique(s)

immunofluorescence: suitable, proximity ligation assay: suitable

suitability

suitable for brightfield

suitability

suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for brightfield, suitable for fluorescence

storage temp.

−20°C

storage temp.

−20°C

storage temp.

20-25°C

storage temp.

−20°C

packaging

pkg of Box A (-20°C)

packaging

-

packaging

-

packaging

-

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

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.

Legal Information

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

Signal Word

Danger

Hazard Classifications

Acute Tox. 3 Dermal - Acute Tox. 4 Inhalation - Acute Tox. 4 Oral - Aquatic Chronic 3 - Eye Dam. 1 - Flam. Liq. 2 - Muta. 2 - Skin Irrit. 2 - Skin Sens. 1

Storage Class Code

3 - Flammable liquids

WGK

WGK 3

Flash Point(F)

42.8 °F

Flash Point(C)

6 °C


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Zhiwei Ang et al.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 32(1), 289-303 (2017-09-09)
Free fatty acid receptors 2 and 3 (FFAR2/FFA2/GPR43 and FFAR3/FFA3/GPR41) are mammalian receptors for gut microbiota-derived short-chain fatty acids (SCFAs). These receptors are promising drug targets for obesity, colitis, colon cancer, asthma, and arthritis. Here, we demonstrate that FFAR2 and
Annabel Christ et al.
Developmental cell, 22(2), 268-278 (2012-02-22)
Sonic hedgehog (SHH) is a regulator of forebrain development that acts through its receptor, patched 1. However, little is known about cellular mechanisms at neurulation, whereby SHH from the prechordal plate governs specification of the rostral diencephalon ventral midline (RDVM)
Agata Zieba et al.
Clinical chemistry, 56(1), 99-110 (2009-11-21)
The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes
Lei Zhu et al.
The Prostate, 73(2), 219-226 (2012-07-19)
PSA is the most useful prostate cancer marker. However, its levels are increased also in some non-malignant conditions. In circulation, the majority of PSA is complexed with protease inhibitors, including α(1) -antichymotrypsin (ACT). The proportion of the PSA-ACT complex is
Shaida Ouladan et al.
International journal of oncology, 46(6), 2595-2605 (2015-04-23)
Solitary fibrous tumors (SFTs) are rare mesenchymal neoplasms, displaying variable morphological and clinicopathological features. Supportive immunohistochemical markers such as CD34, CD99, BCL2 and LSD1 are commonly applied in the differential diagnosis of SFTs, although none is sufficiently sensitive or specific

Articles

How Proximity Ligation Assays (PLA) Work

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.

How to Optimize the Duolink® Proximity Ligation Assay

Things to consider for preparation, setup and execution of the Duolink® assay protocol

Duolink® PLA Troubleshooting Guide

Support information including tips and tricks, frequently asked questions, and basic troubleshooting.

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 tissue and cell samples.

Related Content

Duolink® PLA Applications

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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