Assay Design

Detection and quantification of protein interactions:

When detecting two interacting proteins you need two different primary antibodies, one against each interacting protein. The primary antibodies must be raised in two different species. For a complete Duolink In Situ assay, order one PLA probe PLUS and one PLA probe MINUS against the different species e.g. PLA probe anti-Mouse PLUS and PLA probe anti-rabbit MINUS, together with a Detection Reagents.

Detection and quantification of protein modification (e.g. a phosphorylation):

To be able to detect and quantify a modification, you need one primary antibody against the protein and one primary antibody against the specific modification site, respectively. The two primary antibodies must be raised in two different species. For a complete Duolink In Situ assay, order one PLA probe PLUS and one PLA probe MINUS against the different species, together with a Detection Reagents.

Detection and quantification of a single protein target with high sensitivity:

In order to detect and quantify protein expression with high sensitivity, use only one primary antibody e.g. the same antibody used for immunofluorescense or immunohistochemistry. In this assay, PLA probes PLUS and MINUS are detecting the same primary antibody, which is binding to your protein of interest. Order one PLA probe PLUS and one PLA probe MINUS against the species your primary antibody is raised in, together with a Detection Reagents.

Detection and quantification of a single protein target with high specificity:

This approach is used when you want to detect a single protein target with high specificity and sensitivity. For this application you need two different primary antibodies, raised in two different species against two epitopes on the same protein. For a complete Duolink In Situ assay, order one PLA probe PLUS and one PLA probe MINUS against the different species together with a Detection Reagents.

Create your own PLA probes:

In order to extend the possibilities with in situ PLA technology you can use the Duolink In Situ Probemaker.

Duolink In Situ Probemaker is a product enabling quick and convenient conjugation of the PLA oligo arms directly to primary antibodies. This eliminates the need for PLA probes based on secondary antibodies. Directly conjugating primary antibodies enables same species monoclonal or polyclonal use in a Duolink In Situ assay. As a consequence of this feature it is possible to study homodimeric protein complexes – an ability unique to Duolink.

Simply include Probmaker PLUS and Probemaker MINUS, instead of PLA probes, in a Duolink In Situ kit.