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- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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- Glasbey, JC, et al.
(författare)
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- 2021
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swepub:Mat__t
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| 5. |
- Bravo, L, et al.
(författare)
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- 2021
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swepub:Mat__t
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- Tabiri, S, et al.
(författare)
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- 2021
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swepub:Mat__t
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| 8. |
- Hwang, Hui Yun, et al.
(författare)
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Profiling the Protein Targets of Unmodified Bio-Active Molecules with Drug Affinity Responsive Target Stability and Liquid Chromatography/Tandem Mass Spectrometry
- 2020
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Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 20:9
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Forskningsöversikt (refereegranskat)abstract
- Identifying the target proteins of bioactive small molecules is a key step in understanding mode-of-action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand-modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity-based protein profiling. Target proteins are then enriched by performing a pull-down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read-out). This review highlights recent proteomic approaches utilizing data-dependent analysis and data-independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio-active molecules can be further validated.
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