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- Zieba, Agata, et al.
(författare)
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In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
- 2018
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Ingår i: Modern Pathology. - : Nature Publishing Group. - 0893-3952 .- 1530-0285. ; 31:2, s. 253-263
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.
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| 2. |
- Ebai, Tonge, et al.
(författare)
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Parallel protein detection by solid-phase proximity ligation assay with real-time PCR or sequencing
- 2015
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Ingår i: Current Protocol in Molecular Biology. - : Wiley. - 1934-3647 .- 1934-3639. ; 109:20
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Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assays are a group of protein detection techniques in which reagents with affinity for target proteins, typically antibodies, are coupled to short strands of DNA. DNA-modified affinity reagents are combined in assays constructed such that the coordinated binding of individual target molecules or complexes of interacting proteins by two or more of the reagents, followed by DNA ligation and/or polymerization reactions, gives rise to amplifiable DNA reporter strands. Proximity ligation assays have been shown to exhibit excellent sensitivity in single and multiplexed protein assays for individual or interacting proteins, both in solution and in situ. This unit describes procedures for developing solid-phase proximity ligation assays for soluble proteins using either real-time PCR or DNA sequencing as the readout. In addition, critical steps for assay optimization are discussed.
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