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Proximity Ligation Assay as a Tool for Antibody Validation in Human Tissues.

ABSTRACT: Immunohistochemistry (IHC) is the accepted standard for spatial analysis of protein expression in tissues. IHC is widely used for cancer diagnostics and in basic research. The development of new antibodies to proteins with unknown expression patterns has created a demand for thorough validation. We have applied resources from the Human Protein Atlas project and the Antibody Portal at National Cancer Institute to generate protein expression data for 12 proteins across 39 cancer cell lines and 37 normal human tissue types. The outcome of IHC on consecutive sections from both cell and tissue microarrays using two independent antibodies for each protein was compared with in situ proximity ligation (isPLA), where binding by both antibodies is required to generate detection signals. Semi-quantitative scores from IHC and isPLA were compared with expression of the corresponding 12 transcripts across all cell lines and tissue types. Our results show a more consistent correlation between mRNA levels and isPLA as compared to IHC. The main benefits of isPLA include increased detection specificity and decreased unspecific staining compared to IHC. We conclude that implementing isPLA as a complement to IHC for analysis of protein expression and in antibody validation pipelines can lead to more accurate localization of proteins in tissue.

SUBMITTER: Lindskog C

PROVIDER: S-EPMC7350078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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HiPLA: High-throughput imaging proximity ligation assay.

Project description:Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.

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Proximity ligation assay evaluates IDH1R132H presentation in gliomas.

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| S-EPMC4319432 | biostudies-literature

Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ.

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Analysis of blood group antigens on MUC5AC in mucinous ovarian cancer tissues using in situ proximity ligation assay.

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Tyrosine phosphorylation profiling via in situ proximity ligation assay.

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| S-EPMC4072613 | biostudies-literature

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| S-EPMC4452782 | biostudies-literature

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Project description:Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.

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| S-EPMC4227721 | biostudies-literature

Analysis of close associations of uropod-associated proteins in human T-cells using the proximity ligation assay.

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| S-EPMC3807586 | biostudies-literature

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