Application

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.

Duolink In Situ Probemaker extends your possibilities even further by allowing you to create your own PLA^® probes to meet specific assay requirements.
• Study homodimers
• Study protein-protein interactions with two primary antibodies derived from the same species
• Study protein-protein interactions with primary antibodies from any species

Using Duolink In Situ Probemaker PLUS and MINUS you simply conjugate the PLUS and MINUS oligo arms directly to your antibodies in a quick and convenient procedure. As well as providing unique capabilities, this also means there is no longer any limitation as to which species your primary antibodies have to be derived from in order perform a Duolink In Situ assay.

In order to perform a complete Duolink assay, combine one Duolink In Situ Probemaker PLUS, one Duolink In Situ Probemaker MINUS and your choice of Detection Reagents. Recommended reagents include Wash Buffers and Mounting Medium.

Analysis is carried out using standard immunofluorescence assay equipment. HRP/Novared is also available for bright field detection. Quick and reliable quantification can be performed using Duolink ImageTool.

Application Note
The antibody to be conjugated must have a concentration of 1 mg/ml. Do not use higher volumes than 20 μl for conjugation. The antibody must be in an amine free buffer, ideally PBS. The buffer should be carrier free but may contain up to 0.1% BSA, 5% trehalose and 0.02% sodium azide.
• Study homodimers by using one monoclonal antibody split into two halves. Label one with the PLUS oligo and the other with the MINUS oligo.
• Use antibodies from same species: study protein-protein interactions, protein modifications, or single proteins, using two primary antibodies derived from the same species. Label one of the antibodies with the PLUS oligo and the other with the MINUS oligo
• Use antibodies from any species: study protein-protein interactions, protein modifications, or single proteins, using one or both primary antibodies derived from species other than mouse, rabbit or goat. Label one of your secondary antibodies with the PLUS oligo and the other with the MINUS oligo. Or, combine one labeled secondary antibody with a standard secondary Duolink In Situ PLA probe.

Test your primary antibodies (IgG-class, mono- or polyclonal) in a standard immunofluorescence (IF) or immunohistochemistry (IHC) assay to determine the optimal fixation, blocking, and titer conditions.

Duolink In Situ reagents are suitable for use on fixed cells, cytospin preparations, formalin-fixed paraffin embedded (FFPE) cells or tissue sections, or tissue (fresh or frozen). No minimum number of cells is required.

Features and Benefits

• Quicker than FRET
Visualize protein-protein interactions under endogenous expression
• Increased accuracy compared to co-IP
Single molecule detection capability due to >500X signal amplification
• Greater precision than current immuno assay techniques
Dual binding of primary antibodies enable greater specificity and low false positives

Legal Information

Duolink is a registered trademark of Olink AB

PLA is a registered trademark of Olink AB