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The technique was developed by Fredriksson and colleagues in 2002 and has been shown to overcome the difficulties that arise when attempting to visualize and study single proteins, protein-protein interactions, and post-translational modifications (PTMs), such as phosphorylation, acetylation, and glycosylation.
PLA can be used in advanced protein applications and diverse samples such as biological samples, cell lines, and fresh, frozen or formalin-fixed, paraffin-embedded (FFPE) tissues. It is also a particularly useful technique to elucidate how cells function in health and disease.
Enhanced selectivity for this technique comes from the utilization of a pair of DNA proximity probes, each composed of a specific antibody linked to a high-affinity oligonucleotide (Ab–Oligo), which simultaneously binds to different epitopes of the same protein, or to two proteins in a complex, thereby converting them to DNA molecules for downstream quantification.
Two different methods exist for Proximity Ligation Assay – direct and indirect, but either method uses antibodies raised in different species (see Figure 1.). The direct primary method uses Ab–Oligo conjugates and the indirect method uses unmodified primary antibodies that are detected with secondary Ab–Oligo conjugates. The technique can use either monoclonal or polyclonal antibodies or a combination of both.
Figure 1. Direct and indirect PLA techniques. The direct method uses antibody pairs with primary conjugation and the indirect method uses secondary antibody conjugates.
For either method, additional ‘connector’ oligonucleotides are introduced, which recognize and bind to the free ends of the two Ab–Oligo conjugates, and are subsequently brought into close proximity (30–40nm apart), where they become ligated by DNA ligase.
This enzymatic ligation joins the 3’ end of the first probe with the 5’ end of the second probe, resulting in the formation of a unique target DNA reporter strand (molecule) that is a surrogate marker for the specific protein to be detected, and which contains specific molecular barcodes. These strands participate in rolling circle amplification (RCA) to amplify the product for quantification by real-time PCR.
Homogenous Proximity Ligation Assay
Homogenous Proximity Ligation Assay is suitable for the quantitation of low concentrations of protein molecules in a small homogenous solution, reaching femtomolar detection sensitivity. This method of PLA is performed in three steps, with no wash step required. Briefly, the target antigen is incubated with two proximity probes (3’ and 5’) that bind to adjacent epitopes on the target antigen, and a connector oligonucleotide (~20 bps), which hybridizes to both probes. The DNA molecule generated is amplified, detected, and quantitated by real-time PCR.
As the connector oligo is added in molar excess, free proximity probes are blocked from further hybridization, resulting in low background noise. The added advantage of homogenous PLA is that solid support is not required to immobilize the target protein.
Solid Phase Proximity Ligation Assay
Solid Phase Proximity Ligation Assay is an alternative methodology to homogenous PLA and utilizes a capture antibody to immobilize a target protein onto a solid phase, providing some advantages such as investigation of larger sample sets. The sample is combined with a capture antibody and incubated with the proximity probes. Thus, the target antigen is sandwiched between the proximity probes and the capture antibody.
Ligation and quantitative PCR (qPCR) are conducted following wash steps. One advantage of solid-phase PLA is the elimination of extensive, carefully controlled wash steps, as required for other solid-phase assays such as ELISA. Immobilization of the protein on a solid support can serve to purify and concentrate the samples, especially those that contain constituents that may inhibit ligation or amplification, or where the concentration of analyte is extremely low. The wash step may also remove unbound probes prior to ligation, which would otherwise result in nonspecific probe ligation.
In situ Proximity Ligation Assay
In situ PLA is a third proximity-mediated detection method that can detect and visualize target proteins/protein-protein complexes expressed by fixed cells and on tissue slide sections. As well as using a fixative that is appropriate for the antibodies used in the protocol to fix the cells or tissue, they may sometimes need to be permeabilized. If this step is required, then antigen retrieval and antibody specific blocking must also be performed.
Advantages of Proximity Ligation Assay:
Limitations of Proximity Ligation Assay:
To overcome the complexity of conjugating antibodies to oligonucleotides, we offer an oligonucleotide conjugation kit, an easy to use kit that enables rapid conjugation of antibodies to oligonucleotides, with high recovery of materials and a superior clean-up procedure. The kit is quick and simple to use, overcoming time-consuming and lengthy protocols associated with standard conjugation methods.
Features and Benefits:
Figure 2. The oligonucleotide conjugation process. The Ab–Oligo conjugation protocol consists of three main steps: activation of both the antibody and the oligonucleotide followed by desalting, after which the two are mixed and left to incubate overnight. If an unbound oligonucleotide removal step is necessary for the end application, this can be performed the next morning. Following this, the conjugate is ready to use.
Main applications for the oligonucleotide conjugation kit:
ImmunoPCR (iPCR), Proximity Ligation Assay (PLA), Electrochemical Proximity Assay (ECPA), Lateral Flow.