The volume details techniques, methods, and conceptual developments to further the study of protein aggregation with emphasis on the pleiomorphic proteins implicated in etiology of neurodegeneration. Chapters guide readers through in vitro and in vivo studies of fibrillization and liquid-liquid phase separation processes, and offer a comprehensive account of the state-of-art of structural studies of protein aggregation. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Protein Aggregation: Methods and Protocols aims to be useful and practical guide to new researchers and experts looking to expand their knowledge.
Early aggregation of Amyloid- (1-42) studied by Fluorescence
Correlation Spectroscopy.- Preparation and investigation of crucial oligomers
in the early stages of A40 and A42 aggregation.- Preparation and
fractionation of heterogeneous A42 oligomers with different aggregation
properties.- An efficient method of expression and purification of amyloid
beta (A1-42) peptide from E.coli.- Solid-state NMR structure of amyloid-
fibrils.- Time-resolved in situ AFM measurement of growth rates of A40
fibrils.- Monitoring kinetics of pH-dependent aggregation and disaggregation
of the Pmel17 repeat domain.- Analysis of Tau:nucleoporin interactions by
Surface Plasmon Resonance Spectroscopy.- Microfluidic chamber technology to
study missorting and spreading of Tau protein in Alzheimer disease.- Using
FRET-based biosensor cells to study the seeding activity of tau and
-synuclein.- Functional applications of stable tau oligomers in cell biology
and electrophysiology studies.- An additive-free model for tau
self-assembly.- Cross-linking mass spectrometry analysis of metastable
compact structures in intrinsically disordered proteins.- A validated method
to prepare stable tau oligomers.- Light microscopy and dynamic light
scattering to study liquid-liquid phase separation of Tau proteins in
vitro.- Study of tau liquid-liquid phase separation in vitro.- Liquid-Liquid
Phase Separation to study the association of proteins in solution.- Mapping
phase diagram of tau-RNA LLPS under live cell coculturing conditions.- The
role of buffers in wild-type HEWL amyloid fibril formation mechanism - a
methodological approach.- Reproducible formation of insulin superstructures:
amyloid-like fibrils, spherulites and particulates.- CD and solid-state NMR
studies of low-order oligomers of transthyretin.- Identifying biological and
biophysical features of different maturation states of -synuclein amyloid
fibrils.- Preparation of -synuclein fibril, ribbon and fibril-91 amyloid
polymorphs for structural studies.- Propagation of distinct alpha-synuclein
strains within human reconstructed neuronal network and associated neuronal
dysfunctions.- Single-particle analysis of the interaction between molecules
and protein aggregated species by Dual-Color Time-Resolved Fluorescence
Spectroscopy.- FRAP & FRET investigation of -synuclein fibrillization via
liquid-liquid phase separation in vitro and in HeLa
cells.- Spectrally-resolved FRET microscopy of
-synuclein phase-separated liquid droplets.- Combined H-N
cross polarization and carbonyl detection NMR spectroscopy allows to record
high-resolution, high-sensitivity spectra of alpha-synuclein in bacterial
cells.- Identification of distinct soluble states during fibril formation
using multilinear analysis of NMR diffusiondata.- Structural analysis of SOD1
fibrils with mass spectrometry, limited proteolysis and atomic force
microscopy (AFM).- Biophysical studies of LLPS and aggregation of TDP-43
LCD.- A spectrophotometric turbidity assay to study Liquid-Liquid Phase
Separation of UBQLN2 in vitro.- An optimized SG detection method:
Investigation of UBQLN2 effect on RNA-FUS interaction and SG
formation.- Neuronal puncta/aggregate formation by wild-type and mutant
UBQLN2.- In vivo analysis of a biomolecular condensate in the nervous system
of C. elegans.- FLIM-FRET investigation of heterogeneous huntingtin
aggregation in HeLa cells.- In vitro characterization of protein:nucleic acid
liquid-liquid phase separation by microscopy methods and nanoparticle
tracking analysis.- Cross-seeding assay in the investigation of the amyloid
core of prion fibrils.- Mapping the domain structure and aggregation
propensity of proteins using a Gateway plasmid vector system.
