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Mechanical Fractionation of Cultured Neuronal Cells into Cell Body and Neurite Fractions

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Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells

Authors: Natalia Pacheco-Fernandez, Mehrshad Pakdel and Julia von Blume, date: 03/05/2021, view: 1501, Q&A: 0

[Abstract]

Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling,

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Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS

Authors: Emma Robinson, James Shorter and Lin Guo, date: 08/20/2020, view: 1774, Q&A: 0

[Abstract] The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.

Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues

Authors: Siou Ku, Claire Heichette, Laurence Duchesne and Denis Chrétien, date: 08/20/2020, view: 1306, Q&A: 0

[Abstract] Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, ...

Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus

Authors: Simon Goto, Kotaro Ishida, Ryosuke Suzuki and Eiji Morita, date: 05/05/2020, view: 1514, Q&A: 0

[Abstract] Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such ...

Studying Protein Aggregation in the Context of Liquid-liquid Phase Separation Using Fluorescence and Atomic Force Microscopy, Fluorescence and Turbidity Assays, and FRAP

Authors: W. Michael Babinchak and Witold K. Surewicz, date: 01/20/2020, view: 3705, Q&A: 0

[Abstract] Liquid-liquid phase separation (LLPS) underlies the physiological assembly of many membrane-less organelles throughout the cell. However, dysregulation of LLPS may mediate the formation of pathological aggregates associated with neurodegenerative diseases. Here, we present complementary experimental approaches to study protein aggregation within ...

Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro

Authors: He Sun, Yuanyuan Luo and Yansong Miao, date: 12/05/2018, view: 3465, Q&A: 0

[Abstract] Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable ...

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells

Authors: Archana Varadaraj, Carina Magdaleno and Karthikeyan Mythreye, date: 08/20/2018, view: 3714, Q&A: 0

[Abstract] Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell ...

α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

Authors: Michael M. Wördehoff and Wolfgang Hoyer, date: 07/20/2018, view: 7959, Q&A: 1

[Abstract] Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive ...

Preparation of Amyloid Fibril Networks

Authors: Mirren Charnley , Jay Gilbert, Owen G. Jones and Nicholas P. Reynolds, date: 02/20/2018, view: 5663, Q&A: 0

[Abstract] Networks of amyloid nanofibrils fabricated from common globular proteins such as lysozyme and β-lactoglobulin have material properties that mimic the extracellular microenvironment of many cell types. Cells cultured on such amyloid fibril networks show improved attachment, spreading and in the case of mesenchymal stem cells improved ...

Characterization of Amyloid Fibril Networks by Atomic Force Microscopy

Authors: Mirren Charnley , Jay Gilbert, Owen G. Jones and Nicholas P. Reynolds, date: 02/20/2018, view: 5851, Q&A: 0

[Abstract] Dense networks of amyloid nanofibrils fabricated from common globular proteins adsorbed to solid supports can improve cell adhesion, spreading and differentiation compared to traditional flat, stiff 2D cell culture substrates like Tissue Culture Polystyrene (TCPS). This is due to the fibrous, nanotopographic nature of the amyloid fibril networks ...

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