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@ARTICLE{Amaral:278082,
      author       = {Amaral, Mariana and Passos, Aline R and Mohapatra, Satabdee
                      and Freire, Maria Heloisa and Wegmann, Susanne and Cordeiro,
                      Yraima},
      title        = {{X}-{R}ay {P}hoton {C}orrelation {S}pectroscopy,
                      {M}icroscopy, and {F}luorescence {R}ecovery {A}fter
                      {P}hotobleaching to {S}tudy {P}hase {S}eparation and
                      {L}iquid-to-{S}olid {T}ransition of {P}rion {P}rotein
                      {C}ondensates.},
      journal      = {Bio-protocol},
      volume       = {15},
      number       = {8},
      issn         = {2331-8325},
      address      = {Sunnyvale, CA},
      publisher    = {bio-protocol.org},
      reportid     = {DZNE-2025-00573},
      pages        = {e5277},
      year         = {2025},
      abstract     = {Biomolecular condensates are macromolecular assemblies
                      constituted of proteins that possess intrinsically
                      disordered regions and RNA-binding ability together with
                      nucleic acids. These compartments formed via liquid-liquid
                      phase separation (LLPS) provide spatiotemporal control of
                      crucial cellular processes such as RNA metabolism. The
                      liquid-like state is dynamic and reversible, containing
                      highly diffusible molecules, whereas gel, glass, and solid
                      phases might not be reversible due to the strong
                      intermolecular crosslinks. Neurodegeneration-associated
                      proteins such as the prion protein (PrP) and Tau form
                      liquid-like condensates that transition to gel- or
                      solid-like structures upon genetic mutations and/or
                      persistent cellular stress. Mounting evidence suggests that
                      progression to a less dynamic state underlies the formation
                      of neurotoxic aggregates. Understanding the dynamics of
                      proteins and biomolecules in condensates by measuring their
                      movement in different timescales is indispensable to
                      characterize their material state and assess the kinetics of
                      LLPS. Herein, we describe protein expression in E. coli and
                      purification of full-length mouse recombinant PrP, our in
                      vitro experimental system. Then, we describe a systematic
                      method to analyze the dynamics of protein condensates by
                      X-ray photon correlation spectroscopy (XPCS). We also
                      present fluorescence recovery after photobleaching
                      (FRAP)-optimized protocols to characterize condensates,
                      including in cells. Next, we detail strategies for using
                      fluorescence microscopy to give insights into the folding
                      state of proteins in condensates. Phase-separated systems
                      display non-equilibrium behavior with length scales ranging
                      from nanometers to microns and timescales from microseconds
                      to minutes. XPCS experiments provide unique insights into
                      biomolecular dynamics and condensate fluidity. Using the
                      combination of the three strategies detailed herein enables
                      robust characterization of the biophysical properties and
                      the nature of protein phase-separated states. Key features
                      • For FRAP in cells, we recommend using a spinning disk
                      confocal microscope coupled with temperature and CO2
                      incubator. • For fluorescence microscopy, we recommend
                      simultaneously imaging differential interference contrast
                      (DIC) (or phase contrast) and fluorescence channels to
                      obtain morphological details of phase-separated structures.
                      • For XPCS, coherent X-ray beams, fast X-ray detectors in
                      fourth and third synchrotron light sources, and X-ray
                      free-electron lasers are required.},
      keywords     = {Amyloid dye (Other) / Biomolecular condensates (Other) /
                      Fluorescence microscopy (Other) / Fluorescence recovery
                      after photobleaching (FRAP) (Other) / Liquid-liquid phase
                      separation (LLPS) (Other) / Liquid-to-solid transition
                      (Other) / Phase transitions (Other) / X-ray photon
                      correlation spectroscopy (XPCS) (Other)},
      cin          = {AG Wegmann},
      ddc          = {570},
      cid          = {I:(DE-2719)1810006},
      pnm          = {352 - Disease Mechanisms (POF4-352)},
      pid          = {G:(DE-HGF)POF4-352},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:40291420},
      pmc          = {pmc:PMC12021588},
      doi          = {10.21769/BioProtoc.5277},
      url          = {https://pub.dzne.de/record/278082},
}