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Protocols for Processing and Interpreting cryoEM Data Using Bsoft: A Case Study of the Retinal Adhesion Protein, Retinoschisin

Author: J. Bernard Heymann, date: 01/20/2020, view: 32, Q&A: 0

The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen ...

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: 42, Q&A: 0

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 ...

Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range

Authors: Mikhail Drobizhev, Rosana S Molina and Thomas E Hughes, date: 01/20/2020, view: 39, Q&A: 0

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal ...

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Protocols for Processing and Interpreting cryoEM Data Using Bsoft: A Case Study of the Retinal Adhesion Protein, Retinoschisin

Author: J. Bernard Heymann, date: 01/20/2020, view: 32, Q&A: 0

[Abstract] The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen require adaptions to properly interpret the data. Here I describe the protocols for examining the higher ...

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: 42, 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 ...

Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range

Authors: Mikhail Drobizhev, Rosana S Molina and Thomas E Hughes, date: 01/20/2020, view: 39, Q&A: 0

[Abstract] Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in ...

Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells

Authors: Qadeer Aziz, Muriel Nobles and Andrew Tinker, date: 01/05/2020, view: 124, Q&A: 0

[Abstract] Cardiac pacemaker cells of the sino-atrial node are responsible for the initiation of the heart beat and express an array of ion channels. The patch-clamp technique is the gold standard method for investigating the function of ion channels expressed in electrically active cells. Conventional whole-cell and perforated patch-clamp techniques can be ...

Evoked Potential Recordings of Auditory Brainstem Activity in the Mouse: An Optimized Method for the Assessment of Hearing Function of Mice

Author: Neil J. Ingham, date: 12/05/2019, view: 391, Q&A: 0

[Abstract] Hearing loss is a common sensory deficiency suffered by millions worldwide. It is a heterogeneous condition and genetics plays a critical role in its etiology. Gene variants can fundamentally alter hearing function, or predispose the auditory system towards loss of function resulting from other factors. In mouse studies of hearing loss and gene ...

Opto-magnetic Selection and Isolation of Single Cells

Authors: Loïc Binan, Joannie Roy and Santiago Costantino, date: 11/20/2019, view: 482, Q&A: 0

[Abstract] Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or ...

Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses

Authors: Benji C. Bateman, Laura C. Zanetti-Domingues, Amy N. Moores, Sarah R. Needham, Daniel J. Rolfe, Lin Wang, David T. Clarke and Marisa L. Martin-Fernandez, date: 11/20/2019, view: 741, Q&A: 0

[Abstract] Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to ...

Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion

Authors: Nicola de Franceschi, Maryam Alqabandi, Winfried Weissenhorn and Patricia Bassereau, date: 07/05/2019, view: 889, Q&A: 0

[Abstract] In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on ...

Detection of Ligand-binding to Membrane Proteins by Capacitance Measurements

Authors: Verena Burtscher, Matej Hotka and Walter Sandtner, date: 01/05/2019, view: 2134, Q&A: 0

[Abstract] In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Accordingly, studying ligand-receptor interactions at the cellular ...

Preparation of Silk Fibroin Nanoparticles and Enzyme-Entrapped Silk Fibroin Nanoparticles

Author: Yu-Qing Zhang, date: 12/20/2018, view: 2925, Q&A: 0

[Abstract] After silk fiber is degummed in boiling 0.2% Na₂CO₃ solution, the degummed fibroin fiber could be dissolved in highly concentrated neutral salts such as CaCl₂. The partially degraded polypeptides of silk fibroin, commonly called as regenerated liquid silk, could be obtained via water dialysis. The silk fibroin ...

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