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Non-separate Mouse Sclerochoroid/RPE/Retina Staining and Whole Mount for the Integral Observation of Subretinal Layer

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Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803

Authors: Anabella Aguillera, Charlotte Steelheart, Matías Alegre, Federico Berdun, Graciela Salerno, Carlos Bartoli, Gabriela Pagnussat and María Victoria Martin, date: 10/20/2020, view: 474, Q&A: 0

[Abstract] Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 μM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to ...

In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD

Authors: Ali Camara, Anwesha Sanyal, Sayan Dutta, Jean-Christophe Rochet and Seema Mattoo, date: 09/20/2020, view: 1274, Q&A: 0

[Abstract] One of the major histopathological hallmarks of Parkinson’s disease are Lewy bodies (LBs) –cytoplasmic inclusions, enriched with fibrillar forms of the presynaptic protein alpha-synuclein (α-syn). Progressive deposition of α-syn into LBs is enabled by its propensity to fibrillize into insoluble aggregates. We recently described a marked reduction ...

In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance

Authors: Marina Franceschetti, Mark J. Banfield, Clare E. M. Stevenson and Juan Carlos De la Concepcion, date: 07/05/2020, view: 1498, Q&A: 0

[Abstract] The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...

Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation

Authors: Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 1302, Q&A: 0

[Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...

Fibrin Breakdown Assay

Authors: Jiayue Ling, Connor M. Blair and George S. Baillie, date: 04/20/2020, view: 1773, Q&A: 0

[Abstract] Fibrinolysis is an integral part of the matrix remodeling process that contributes to tissue repair. Fibrin clots are broken down during fibrinolysis in a controlled process. Fibrin degradation products (FDPs) have also been shown to have a role in the regulation of cell growth and are implicated in various vascular diseases. This protocol was ...

Quantitative Determination of Ca²⁺-binding to Ca²⁺-sensor Proteins by Isothermal Titration Calorimetry

Authors: Seher Abbas and Karl-Wilhelm Koch, date: 04/05/2020, view: 1566, Q&A: 0

[Abstract] Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from ...

Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method

Authors: Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 1455, Q&A: 0

[Abstract] This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (E_m = -55 mV) in the SdhA flavoprotein subunit of succinate ...

Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry

Authors: Claudia Cattoglio, Xavier Darzacq, Robert Tjian and Anders Sejr Hansen, date: 02/20/2020, view: 2481, Q&A: 0

[Abstract] Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence ...

Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces

Authors: Roberta M Sabino and Ketul C Popat, date: 02/05/2020, view: 2124, Q&A: 0

[Abstract] Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. ...

Semi-quantitative Determination of Protein Expression Using Immunohistochemistry Staining and Analysis: An Integrated Protocol

Authors: Alexandra R Crowe and Wei Yue, date: 12/20/2019, view: 3569, Q&A: 0

[Abstract] Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed ...

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