Proximity Ligation Assay

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The Proximity Ligation Assay (PLA) is an in situ application similar to immunohistochemistry (IHC) or immunocytochemistry (ICC). As with those classic in situ assays, tissues or cells may be processed on microscope slides; however, PLA also allows for processing samples in 96-well TC plates (for cell-based assays), which makes it conducive for performing high-throughput screens. The current form of PLA relies on the use of commercially available reagent sets for the conjugation, ligation, and amplification parts of the protocol. The basic principle of PLA is the same as IHC/ICC in that a primary antibody is used that recognizes a specific protein. After binding the epitope recognized by the primary antibody, detection is performed that allows visualization of the protein target so that it can be localized to a particular tissue and/or cellular organelle. In PLA, detection can be done via direct (primary-conjugate) or indirect (secondary-conjugate) methods, just as with IHC/ICC. In PLA, each of two antibodies is conjugated to different oligonucleotides, which are used to generate a ligation product that enables rolling circle amplification (RCA). Detection of the antibody pair is then done by the hybridization of a labeled oligonucleotide that binds the amplification product, i.e., it is basically an in situ southern blot. The resulting imaging is very clean, and the signal strength is amplified as much as 500-fold above that for typical IHC/ICC.

 

PLA in HeLa cells. Goat anti-human RFC1 (A300-141A) and rabbit anti-human BRD4 (A301-985A100). Image merged from DAPI (2ms) and Texas Red (200ms) exposures, 40X magnification.

Goat anti-human RFC1 (A300-141A), negative control with rabbit IgG.

Rabbit anti-human BRD4 (A301-985A100), negative control with goat IgG.

Below is a complete listing of Bethyl antibodies that are validated for use in PLA:

 

PLA