Abstract
Protein-protein interaction experiments, such as co-immunoprecipitation (IP) assays, classically require tremendous amount of cells. This becomes a problem when your work focuses on rare cell populations (e.g., lymphocyte subtypes). O-link Bioscience has developed Proximity Ligation Assay (PLA) reagents and procedures to alleviate and solve this kind of issue. Moreover PLA experiments are read out using fluorescence or bright field microscopy, providing additional information on intracellular interactions localization significantly bettering classical IP procedures.PLA reagents are made of complementary small oligonucleotides “minus” and “plus” probes which specifically recognize host species from the primary antibodies (Abs) targeting the two proteins you are interested in. Experiments have to be designed with primary Abs from different species (rabbit, mouse or goat) as PLA probes “minus” or “plus” react against a specific host species of the primary antibody (e.g. “plus” anti-rabbit with “minus” anti-mouse or “plus” anti-mouse with “minus” anti-rabbit combos are allowed if mouse and rabbit primary Abs are used). When the two PLA probes are close enough (40 nm) a ligation occurs upon ligase incubation generating a circle DNA. These circle-forming DNAs are next amplified thanks to a polymerase and complementary fluorescent nucleotides, being incorporated at this step. Each luminescent spot is thereafter considered being an interaction site between the two proteins (Figure 1).Figure 1. Schematic representation of Duolink® experiment. Primary Abs from different host species were used [i.e., Mouse (Ms) anti-Nlrp3 and Rabbit (Rb) anti-IRF4]. When protein-protein interaction occurs PLA probes allow incorporation of fluorescent oligonucleotides which are analyzed by microscopy.PLA experiments have been performed on differentiated T cells from mice. T cells were grown on cover slip coated with poly-L-Lysine. Please be aware that for T cells and other non-adherent cells, experiments and staining are also possible in 500 µl microtubes until the last washing step before mounting cover slip on slide. This microtube method saves cytokines reagents and allows T cells to grow with usual methods but centrifugation repetition for washing steps is hazardous. Primary Abs incubation also requires also a specific setup if microtube method is chosen.
Keywords: Protein interaction, Fluorescence, CD4 T cells
Materials and Reagents
Equipment
Software
Procedure
Representative data
Figure 3. Reprensative images for Duolink spots in TH2 cells. T cells pictures obtained with our protocol (Nv: Naïve, Scale bar in yellow: 5 µm). Red dots in Th2 cells are Duolink spots.
Notes
Acknowledgments
This work was supported by a French Government grant managed by the French National Research Agency under the program “Investissements d’Avenir” with reference ANR-11-LABX-0021 (Lipstic Labex). FG team is « Equipe labélisée Ligue Nationale Contre le Cancer ». V. D. is the recipient of a « poste d’accueil INSERM »; This protocol was adapted from previous work of Fredriksson et al. (2007) and Lin et al. (2015).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.