Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes

Abstract

BackgroundGenetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy.MethodsWe describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm.ResultsWe propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells.ConclusionThe optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-017-0328-2) contains supplementary material, which is available to authorized users.

Limited significance of the in situ proximity ligation assay

“…The standard negative control of the isPLA method, i.e. simple omission of one of the two primary antibodies [6,9,16], is insufficient, as it only controls for the potential non-specific binding of the secondary antibodies (and here PLA probes), which is generally very low anyway.…”

“…The potential applications of this method are huge, since it can be used in principle with any pair of antibodies, allowing co-detection of any endogenous antigen, including posttranslational modifications, such as specific phosphorylated sites. This flexibility explains its increasing popularity, in fields as diverse as cell biology, pharmacology, immunology, virology, proteomics, biomarkers for cancer, pathogen diagnostic, or even astrobiology [5,7,8,[10][11][12][13][14][15][16][17].…”

CaMKIIδ interacts directly with IKKβ and modulates NF-κB signalling in adult cardiac fibroblasts

“…The Duolink® in situ Orange Proximity Ligation Assay (PLA) was used to detect CaMKII-IKK interactions in CFs following manufacturers' protocol (Sigma-Aldrich) and as previously described [20]. Optimisation of antibodies was performed initially by immunofluorescence (IKK , 1:β00, mouse monoclonal (Abcam); CaMKII , 1:50, rabbit polyclonal (Eurogentec custom-made); ox-CaMKII, 1:50, rabbit polyclonal (GeneTex).…”

Roles of developmentally regulated KIF2A alternative isoforms in cortical neuron migration and differentiation

“…Fig.3). Next, we confirmed the interaction between KIF2A and RTN4 by proximity ligation assay (PLA), which is a sensitive and specific method for detecting protein interactions with spatial resolution 37 . Our PLA results revealed a significant interaction between KIF2A and RTN4, compared to negative controls where only anti-KIF2A or anti-RTN4 primary antibodies were used for the PLA protocol ( Fig.5B and C).…”