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DUO92014

Duolink® In Situ Detection Reagents Green

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About This Item

NACRES:
NA.32
UNSPSC Code:
12352200

$660.00

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product line

Duolink®

Quality Level

200

technique(s)

proximity ligation assay: suitable

fluorescence

λex 495 nm; λem 527 nm (green) (FITC (Cyanine 2), Zeiss Filter set 38)

suitability

suitable for fluorescence

shipped in

dry ice

storage temp.

−20°C

General description

Duolink® In Situ Detection Reagents Green 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 = 495 nm and lem = 527 nm), which may be visualized using the same filter as Cy®2 or FITC. 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® In Situ Detection Reagents has been used in the proximity ligation assay of:
  • vasopressin and gonadotropin-releasing hormone (GnRH) from frozen rat brain sections[1]
  • human embryonic kidney 293 cells (HEK)[2]
  • pituitary tissues[3]

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.[4][5][6]

Biochem/physiol Actions

Green fluorescence detection reagents are often used with FITC filter.

Features and Benefits

  • No overexpression or genetic manipulation reNo 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

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.
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.

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

View full Duolink® product list

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

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Duolink® In Situ Detection Reagents Green

Sigma-Aldrich

DUO92014

Duolink® In Situ Detection Reagents Green

Duolink® In Situ Detection Reagents FarRed

Sigma-Aldrich

DUO92013

Duolink® In Situ Detection Reagents FarRed

Duolink® In Situ Detection Reagents Orange

Sigma-Aldrich

DUO92007

Duolink® In Situ Detection Reagents Orange

Duolink® In Situ Detection Reagents Red

Sigma-Aldrich

DUO92008

Duolink® In Situ Detection Reagents Red

technique(s)

proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

technique(s)

proximity ligation assay: suitable

suitability

suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for fluorescence

fluorescence

λex 495 nm; λem 527 nm (green) (FITC (Cyanine 2), Zeiss Filter set 38)

fluorescence

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

fluorescence

λex 554 nm; λem 576 nm (Cyanine 3; Zeiss Filter set 20)

fluorescence

λex 594 nm; λem 624 nm (Texas Red®, Zeiss Filter set 31)

storage temp.

−20°C

storage temp.

−20°C

storage temp.

−20°C

storage temp.

−20°C

shipped in

dry ice

shipped in

dry ice

shipped in

dry ice

shipped in

dry ice

Quality Level

200

Quality Level

200

Quality Level

200

Quality Level

200

signalword

Danger

Storage Class

10 - Combustible liquids

wgk

WGK 3

pictograms

Health hazardEnvironment
GHS08,GHS09

Hazard Classifications

Aquatic Chronic 2 - ED ENV 1 - Resp. Sens. 1

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Certificates of Analysis (COA)

Lot/Batch Number
0000500853
0000500853
0000444292
0000444292
0000444139

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Protocols

Duolink® PLA Fluorescence Protocol

This protocol describes how to perform immunofluorescent detection of proteins in cells and tissue.

Duolink® In Situ Short Instructions - Fluorescence

This page details the Duolink® In Situ Short Protocol for fluorescence detection

Protocole Duolink® PLA en fluorescence

Ce protocole décrit comment réaliser une détection des protéines par immunofluorescence dans les cellules et les tissus.

Articles

Duolink® PLA Troubleshooting Guide

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

How to Optimize the Duolink® Proximity Ligation Assay

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

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

How to Automate Duolink® Assays on the AAW™ Workstation

View an automated protocol for Duolink® assays on the AAW™ automated assay workstation and see results comparing manual vs automated runs.

Visualizing DNA Methylation (5mC) on a Specific Genomic Locus (SEPTIN 9) in Individual Human Cancer Cells

Application Note

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Global Trade Item Number

SKUGTIN
DUO92014-100RXN04061833592175
DUO92014-30RXN04061833592182

Questions

1–3 of 3 Questions  

  1. What are the absolute (or relative) brightness compared with other fluorophores? Brightness of red vs orange vs green vs FarRed for fluorescent microscope imaging (not flow cytometry).

    1 answer

    1. The specific fluorophores used in Duolink kits are proprietary. However, the detection wavelengths and reference fluorophores for filter selection are provided as follows:

      Green: 495/527 nm (Cy2/FITC)
      Orange: 554/579 nm (Cy3)
      Red: 594/624 nm (Texas Red)
      Far Red: 644/669 nm (Cy5)
      Violet: 390/476 nm (excited with 405 nm laser)

      No absolute brightness values are available, but these references allow for relative comparison with standard fluorophores.

      Helpful?

  2. What are the species of the positive and negative probes?

    1 answer

    1. The available probes include:
      Duolink® In Situ PLA® Probe Anti-Rabbit MINUS (Product number: DUO92005)
      Duolink® In Situ PLA® Probe Anti-Rabbit PLUS (Product number: DUO92002)
      Duolink® In Situ PLA® Probe Anti-Mouse MINUS (Product number: DUO92004)
      Duolink® In Situ PLA® Probe Anti-Mouse PLUS (Product number: DUO92001)
      Duolink® In Situ PLA® Probe Anti-Human MINUS (Product number: DUO92021)
      Duolink® In Situ PLA® Probe Anti-Human PLUS (Product number: DUO92020)
      Duolink® In Situ PLA® Probe Anti-Goat MINUS (Product number: DUO92006)
      Duolink® In Situ PLA® Probe Anti-Goat PLUS (Product number: DUO92003)

      Helpful?

  3. Is there a variation in the intensity of the fluorophores recommended in the flow kits, where one may appear brighter compared to the others?

    1 answer

    1. Duolink Fluorophores vary in intensity. Violet is the strongest with low background. Far red is equally strong but may have higher diffuse background in certain cell types. Refer to attached pictures for visual comparison. When doing multicolor flow cytometry, choose fluorophores with minimal spectral overlap. Reserve brightest fluorophore for lowest expressing signal.

      Helpful?

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