A wealth of tools are available today to study cell-signaling mechanisms. When it comes to actually quantifying transient protein-protein interactions within a single native cell, however, adequate tools are missing. Currently used techniques lyse significant quantities of the studied cells and then perform assays such as the classical co-immunoprecipitation (Co-IP) followed by Western blotting- based detection.
Western blotting, a cellular biology workhorse, has been extensively used with great success for many years, but is limited in some essential respects. For example, weak and transient interactions may not survive the washing procedures involved, and the technique consumes large amounts of cultured cells that are difficult to obtain for certain cell types such as stem cells and clinical tissue material. Also, cell lysis does not maintain the subcellular localization of these signaling events.
Standard in situ based protein-detection protocols such as immunofluorescence and immunohistochemistry involve the use of only one single primary antibody recognizing its target protein. In order to enable the study of two different interacting proteins, one would need to devise an assay using two different primary antibodies.
To address this issue, Olink Bioscience released Duolink®, a kit series enabling the use of two primary antibodies for in situ immunoassays, thereby allowing the dual recognition of fixed cells and tissue samples. Historically, dual recognition by two antibodies exemplified by sandwich immunoassays such as ELISA, has drastically enhanced the sensitivity, specificity, and ability to study protein interactions.
Duolink, a high-resolution colocalization technology with a fluorescent spot readout, can be compared to techniques such as fluorescence resonance energy transfer and double-label colocalization.
Use of Duolink requires that users apply two primary antibodies to the cells grown on microscopy slides. These primary antibodies are selected from two different host species such as one mouse and one rabbit antibody. Subsequently, generic secondary antispecies antibodies provided in the Duolink kit are added.
These secondary antibodies are equipped with DNA strands capable of generating a highly fluorescent and localized amplification product when in close proximity (<40 nm)—hence the name in situ Proximity Ligation Assay (PLA). The resulting readout is measured by fluorescence microscopy by counting the number of bright fluorescent spots generated from single molecules of protein-protein interaction events (Figure 1).
The Duolink reagent kit series can be applied to numerous applications but is mainly used for protein-protein interaction analyses. Many scientists studying protein-protein interactions use Co-IP first then apply Duolink and in situ PLA for orthogonal validation, especially when validating their interaction in archived tissue microarray material, where Co-IP is not applicable.
When investigating signaling pathways and protein interactions, being able to specifically stimulate the cells to promote the interaction adds additional assurance to the validity of the results. Figure 2 displays an example of TGF-β stimulation of MEF cells promoting SMAD dimerization.
Upon such stimulation, users may also add substances to disrupt protein-protein interactions and study their respective efficiency by utilizing the quantitative ability of in situ PLA when counting the number of signals per cell. These substances may be monoclonal antibodies in therapeutic development targeting extracellular receptors or small molecule drug candidates.
Single proteins have long been used as diagnostic markers in cancer tissue samples such as HER2 detection for Herceptin treatment guidance. The interaction between HER2 and HER3 in breast cancer tissue as visualized by Duolink is shown in Figure 3.
Protein-protein interactions within signal-transduction pathways show great promise as a new class of biomarkers. Such markers could be used to predict and monitor drug response, particularly in cancer where signaling-pathway disruption can lead to uncontrolled growth. The Duolink reagents provide an efficient way to study interaction-based putative biomarkers at high throughput in archival tissue microarrays.
In summary, the Duolink reagent series provides a powerful yet simple means to quantitatively visualize even weak or transient protein interactions in fixed cells and tissue with high specificity and sensitivity. These generic secondary reagents can be used for multiple species of primary antibodies to fit the unique application of the end user.