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Table 2 Methods to evaluate and quantify protein assemblies

From: Protein assembly systems in natural and synthetic biology

In vitro:
• The canonical approach to determine the amyloid nature of a protein aggregate is by staining with dyes, such as Thioflavin-T (ThT) and Congo Red, which selectively intercalate into the fibril and emit fluorescence at specific wavelengths in a quantitative fashion [100]. ThT-based assays can measure kinetics of aggregation in high throughput [28, 101, 102].
• Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) characterizes the size distribution of large, detergent-resistant aggregates in cell lysates [103].
• X-ray diffraction identifies the symmetry patterns of amyloid fibrils [104].
• Solid-state nuclear magnetic resonance (ssNMR) is used to derive the structural properties of fibrils [105,106,107]. Solution NMR is used to elucidate the early stage dynamics of aggregate formation [108, 109], and the dynamics of conformational changes and interactions with other proteins [110].
• Super-resolution microscopy techniques, such as PALM and STORM, have revealed the morphology of fibers [111] and cryo-electron microscopy has recently produced high-resolution images of protein aggregates [112, 113], all in fixed cells.
In vivo:
• Dynamic properties of protein assemblies can be studied with microscopy techniques, such as fluorescence recovery after photobleaching (FRAP) [114] and Forster resonance energy transfer (FRET) [32].
• DAmFRET enables the determination of nucleation barriers of protein assemblies in living cells [32].
• Super-resolution microscopy (PALM and STORM) has been applied to living cells to measure the growth of amyloid fibrils [115, 116] and visualize the nucleation process [33].
• yTRAP (yeast Transcriptional Reporting of Aggregating Protein) is a genetic system enabling high-throughput sensing and control of protein aggregation states in yeast cells [117].
• A generic sensor of protein aggregation in mammalian cells uses a fusion of HSP27 and GFP [118].
• Split protein systems, such as split TetR in mammalian cells [119] and a tripartite β-lactamase in Escherichia coli [120], enable detection of the solubility of a specific protein.
• Various phenotypic assays have been developed to detect prions by linking prion state with a growth phenotype or reporter in yeast [54, 68, 72, 121,122,123,124].
• Transmissibility of bona fide prions is evaluated using cytoduction in yeast [125].