Proximity Ligation Assay for Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ.
Curr Protoc Cell Biol
; 89(1): e115, 2020 12.
Article
em En
| MEDLINE
| ID: mdl-33044803
ABSTRACT
Biochemical methods can reveal stable protein-protein interactions occurring within cells, but the ability to observe transient events and to visualize the subcellular localization of protein-protein interactions in cells and tissues in situ provides important additional information. The Proximity Ligation Assay® (PLA) offers the opportunity to visualize the subcellular location of such interactions at endogenous protein levels, provided that the probes that recognize the target proteins are within 40 nm. This sensitive technique not only elucidates protein-protein interactions, but also can reveal post-translational protein modifications. The technique is useful even in cases where material is limited, such as when paraffin-embedded clinical specimens are the only available material, as well as after experimental intervention in 2D and 3D model systems. Here we describe the basic protocol for using the commercially available Proximity Ligation Assay™ materials (Sigma-Aldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. © 2020 Wiley Periodicals LLC. Basic Protocol 1 Proximity ligation assay Support Protocol 1 Antigen retrieval method for formalin-fixed, paraffin-embedded tissues Support Protocol 2 Creation of custom PLA probes using the Duolink™ In Situ Probemaker Kit when commercially available probes are not suitable Basic Protocol 2 Imaging, quantification, and analysis of PLA signals.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Especificidade de Órgãos
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Bioensaio
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Células
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Processamento de Proteína Pós-Traducional
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Mapeamento de Interação de Proteínas
Tipo de estudo:
Guideline
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article