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| 2. |
- Al-Jubair, Tamim, et al.
(author)
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Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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Journal article (peer-reviewed)abstract
- Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).
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| 3. |
- Al-Jubair, Tamim, et al.
(author)
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High-yield overproduction and purification of human aquaporins from Pichia pastoris
- 2022
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In: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).
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| 4. |
- Arellano-Caicedo, Carlos, et al.
(author)
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Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models
- 2024
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In: STAR Protocols. - 2666-1667. ; 5:1
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Journal article (peer-reviewed)abstract
- Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1
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| 6. |
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| 7. |
- Bojmar, Linda, 1983-, et al.
(author)
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Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids
- 2021
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In: STAR protocols. - : Cell Press. - 2666-1667. ; 2:1
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Journal article (peer-reviewed)abstract
- We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
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| 8. |
- Bojmar, Linda, et al.
(author)
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Protocol for cross-platform characterization of human and murine extracellular vesicles and particles
- 2024
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In: STAR PROTOCOLS. - : ELSEVIER. - 2666-1667. ; 5:1
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Journal article (peer-reviewed)abstract
- Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross -validation across different platforms.For complete details on the use and execution of this protocol, please refer to Hoshino et al.1
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| 9. |
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| 10. |
- Boutet-Robinet, Elisa, et al.
(author)
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Detection of DNA damage by alkaline comet assay in mouse colonic mucosa
- 2021
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In: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
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Journal article (peer-reviewed)abstract
- We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
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