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 sectionshuman embryonic kidney 293 cells (HEK)pituitary tissues

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.

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 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

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

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.

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

Other Notes

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^® projectsView full Duolink^® product list

Preparation Note

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

Green fluorescence detection reagents are often used with FITC filter.

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.