Specificity
Fluorescence detection reagents are often used with FITC, Cy^®3,Texas Red^® or Cy^®5 filter. The filter chosen is dependent on the color of the Multicolor Probemaker kit selected.

Application Note
Primary antibodies 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.

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Multicolor Duolink^® proximity ligation assay (PLA^®) is an application of the Duolink^® technology that allows for endogenous, simultaneous detection of up to 4 protein interactions, post-translational modifications, or protein expression levels (including low abundant protein) in fixed cells and tissue samples. Follow the Multicolor Duolink PLA protocol to use this product.

• Visualize and detect multiple proteins events in one experiment.
• No overexpression or genetic manipulation required
• High specificity (fewer false positives)
• Detection of low abundant proteins due to rolling circle amplification
• Relative quantification possible
• No special equipment needed
• Faster and more simple than FRET
• Increased accuracy compared to co-IP
• Publication-ready results

Components
Duolink^® PLA Multicolor Reagent Pack contains all the necessary reagents to perform the amplification and detection of bound custom PLA probes generated using Duolink^® PLA Multicolor Probemaker Kits. Sufficient components are provided for the indicated number of reactions (30 or 100 RXN), based on 40 mL of the total reaction mixture covering 1 cm².

• This product is comprised of the following:
• 5X Multicolor PLA LIgation Buffer DUO86002
• Ligase DUO82027 or DUO82025
• 5X Amplification Buffer DUO82050
• Polymerase DUO82028 or DUO82026
• 5X Multicolor PLA Detection Buffer DUO86001
• 1X Blocking Buffer DUO82007
• 1X Probemaker PLA Probe Diluent DUO82036

See datasheet for more information.

To perform a Multicolor Duolink^® PLA experiment you will need primary antibodies (PLA, IHC, ICC or IF validated) that recognize target epitopes. You will also need at least two pair of the resulting Duolink^® PLA Multicolor Probemaker-generated probes (Red A/Red B, Green C/Green D, Orange F/Orange G, and/or FarRed H/FarRed I).The reagents in the Duolink^® PLA Multicolor Reagent Pack are required to perform the amplification and detection of bound Multicolor PLA probes. Recommended additional reagents include Duolink^® Wash Buffers and Mounting Medium.

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

Texas Red is a registered trademark of Life Technologies