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- Cane, Gaëlle, et al.
(författare)
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Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplification for Improved Protein Analyses
- 2017. - 3
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Ingår i: Molecular Diagnostics (Third Edition). - 2016 : Academia Press. - 9780128029718 ; , s. 219-231
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Bokkapitel (refereegranskat)abstract
- Proximity ligation assay (PLA) is a unique method in which single-stranded oligonucleotides are conjugated to affinity binders of proteins, followed by amplification of the signal by DNA polymerization and hybridization of complementary oligonucleotides labeled with fluorogenic or chromogenic readout. Here, a brief overview of the field of protein analysis describes the background and the initial development of the technique for the detection of protein–protein interactions via the proximity probes mentioned. In this context, PLA can constrain the general problem of cross-reactivity in protein detection by affinity binders, by ensuring that only cognate pairs of proximity probes result in a signal. Thereafter, this chapter deals mainly with derivatives methods and their applications, with a particular interest in improved specificity, application to various biological materials, and multiplexing. The method has been applied in situ and in solution, adapted for the detection of posttranslational modifications such as phosphorylation and interactions between proteins and specific DNA sequences, and multiplexed to a certain extent, which illustrates its versatility. A technique free from enzymatic reaction, the hybridization chain reaction, can be considered a cost-effective alternative particularly suitable to molecular diagnostics. Finally, we explore further development toward higher-level multiplexing and sensitivity. At this point it is not clear what level can be achieved by PLA, but the assay is compatible with a wide range of readout, including separate real-time amplification reactions and novel microfluidic read-out platforms.
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| 2. |
- Johansson Wensman, Jonas, et al.
(författare)
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Visualization of Borna Disease Virus Protein Interactions with Host Proteins using in situ Proximity Ligation Assay
- 2016
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Ingår i: British journal of virology. - : ResearchersLinks Ltd. - 2055-6128. ; 3:1, s. 11-23
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Tidskriftsartikel (refereegranskat)abstract
- Borna disease virus type 1 (BDV) comprises highly conserved neurotropic non-segmented negative strand RNA-virus variants causing neurological and behavioral disorders in a wide range of mammalian animals, possibly including humans. Viral persistence in the brain has been frequently observed, however, the exact mechanisms behind BDV’s ability to establish persistence despite a prominent immune response are not known. Here we have used in situ proximity ligation assay (in situ PLA), a selective tool for studying virus-host protein-protein interactions. BDV P (phosphoprotein) and N (nucleoprotein) have previously been reported to interact with several host proteins, thereby interfering with various signaling pathways. In this study, we focused on some of these interactions (BDV P-HMGB1, BDV N/P-Cdc2). First, we used rat glioma cell cultures persistently infected with a laboratory strain of BDV (C6BV) to establish the assay. Next, in situ PLA was applied to detect BDV P in brain tissues of infected animals. Finally, protein-protein interactions were visualized in both C6BV and brain tissues of experimentally as well as naturally infected animals (rat and horse, respectively). BDV proteins and their interactions with host proteins could be shown in cell cultures (HMGB1, Cdc2) and in brain tissues of rat (HMGB1, Cdc2) and horse (Cdc2 only) infected with BDV. In this study, we have for the first time directly visualized protein-protein interactions between BDV and its host, and thereby confirmed previous data to demonstrate findings in cell cultures to be applicable also in experimentally and naturally infected animals.
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