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Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice

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Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis

Authors: Sujeet Kumar and Natividad Ruiz , date: 06/20/2019, view: 286, Q&A: 0

[Abstract] Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of ...

Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool

Authors: Felix Boos, Timo Mühlhaus and Johannes M. Herrmann, date: 09/05/2018, view: 1521, Q&A: 0

[Abstract] Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through ...

Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method

Authors: Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 1461, Q&A: 0

[Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System

Authors: Kuancheng Liu and Jianming Hu, date: 04/20/2018, view: 2142, Q&A: 0

[Abstract] The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for ...

Hydrogen Deuterium Exchange Mass Spectrometry of Oxygen Sensitive Proteins

Authors: Luke Berry, Angela Patterson, Natasha Pence, John W. Peters and Brian Bothner, date: 03/20/2018, view: 3436, Q&A: 0

[Abstract] The protocol detailed here describes a way to perform hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) on oxygen sensitive proteins. HDX-MS is a powerful tool for studying the protein structure-function relationship. Applying this technique to anaerobic proteins provides insight into the mechanism of proteins that perform oxygen ...

Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae

Authors: Kliment A. Verba and David A. Agard, date: 10/05/2017, view: 3931, Q&A: 0

[Abstract] Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of ...

Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells

Authors: Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 5026, Q&A: 0

[Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...

Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography

Authors: Juan Andres Imelio, Nicole Larrieux, Ariel Edgardo Mechaly, Felipe Trajtenberg and Alejandro Buschiazzo, date: 08/20/2017, view: 3330, Q&A: 0

[Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR ...

Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth

Authors: Natalia V. Andreeva and Alexander V. Belyavsky, date: 07/05/2017, view: 4051, Q&A: 0

[Abstract] Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins ...

Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor

Authors: Rajan Lamichhane, Jeffrey J. Liu, Raymond F. Pauszek III and David P. Millar, date: 06/20/2017, view: 4430, Q&A: 0

[Abstract] Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 ...

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