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.

This product can be applied to both the Duolink^® In Situ Fluorescence Protocol and the Duolink^® In Situ Brightfield Protocol depending on the detection reagents used.

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.HRP is also available for brightfield detection.

Specificity
PLA probe anti-Rabbit reacts with whole molecule rabbit IgG and the light chains of other rabbit immunoglobulin?s. The PLA probe anti-Rabbit has minimal cross reactivity with bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, mouse, rat, and sheep serum proteins. A PLUS probe of a different species must be used simultaneously with this product. See our Product Selection Guide for more information.

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^® PLA 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.

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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:

5x PLA Probe Anti-Rabbit MINUS - Donkey anti-rabbit secondary antibody conjugated to oligonucleotide MINUS
1x Blocking Solution - Reagent for blocking of the sample
1x Antibody Diluent - For dilution of PLA probes and primary antibodies

See datasheet for more information.