Molecular Biology

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    Protocols in Current Issue
    Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres
    Authors:  Szilárd Szikora, Tibor Novák, Tamás Gajdos, Miklós Erdélyi and József Mihály, date: 06/20/2020, view: 673, Q&A: 0
    [Abstract] Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful ...
    Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease
    Authors:  Yibing Deng, Shengchao Shi, Bin Zheng, Tao Wu and Peng Zheng, date: 04/20/2020, view: 1304, Q&A: 0
    [Abstract] The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the ...
    Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
    Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 998, 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 (Em = -55 mV) in the SdhA flavoprotein subunit of succinate ...
    Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
    Authors:  Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 1020, Q&A: 0
    [Abstract] High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol ...
    Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network
    Authors:  Xiao Tang, Feng Yang and Yusong Guo, date: 03/05/2020, view: 1057, Q&A: 0
    [Abstract] Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. ...
    A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae
    [Abstract] We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using ...
    SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
    Authors:  Manuela Pérez-Berlanga, Florent Laferrière and Magdalini Polymenidou, date: 11/20/2019, view: 1939, Q&A: 0
    [Abstract] TDP-43 is the main aggregating protein in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Aggregated TDP-43 is resistant to diverse detergent solubilization, yet physiological TDP-43 and other abundant proteins commonly co-purify with pathological TDP-43. This mixed isolation has ...
    Platelet Isolation and Activation Assays
    Authors:  Laura C. Burzynski, Nicholas Pugh and Murray C.H. Clarke, date: 10/20/2019, view: 2369, Q&A: 0
    [Abstract] Platelets regulate hemostasis and are the key determinants of pathogenic thrombosis following atherosclerotic plaque rupture. Platelets circulate in an inactive state, but become activated in response to damage to the endothelium, which exposes thrombogenic material such as collagen to the blood flow. Activation results in a number of responses, ...
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 2451, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...
    Purification and Proteomic Analysis of Alphavirus Particles from Sindbis Virus Grown in Mammalian and Insect Cells
    Authors:  Raquel Hernandez, Trevor Glaros, Gabrielle Rizzo and Davis F. Ferreira, date: 05/20/2019, view: 2561, Q&A: 0
    [Abstract] Current mass spectrometry (MS) methods and new instrumentation now allow for more accurate identification of proteins in low abundance than previous protein fractionation and identification methods. It was of interest if this method could serve to define the virus proteome of a membrane-containing virus. To evaluate the efficacy of mass spec to ...



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