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    Protocols in Current Issue
    Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae
    Authors:  Laura E. Knighton, Lizbeth P. Saa, Adam M. Reitzel and Andrew W. Truman, date: 10/05/2019, view: 292, Q&A: 0
    [Abstract] Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as Nematostella ...
    Yeast Single-cell RNA-seq, Cell by Cell and Step by Step
    [Abstract] Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast ...
    Non-invasive Quantification of Cell Wall Porosity by Fluorescence Quenching Microscopy
    Authors:  Xiaohui Liu, Thomas Günther Pomorski and Johannes Liesche, date: 08/20/2019, view: 505, Q&A: 0
    [Abstract] All bacteria, fungi and plant cells are surrounded by a cell wall. This complex network of polysaccharides and glycoproteins provides mechanical support, defines cell shape, controls cell growth and influences the exchange of substances between the cell and its surroundings. Despite its name, the cell wall is a flexible, dynamic structure. ...
    QUEEN-based Spatiotemporal ATP Imaging in Budding and Fission Yeast
    Author:  Masak Takaine, date: 08/05/2019, view: 719, Q&A: 0
    [Abstract] Yeasts have provided an exceptional model for studying metabolism and bioenergetics in eukaryotic cells. Among numerous metabolites, adenosine triphosphate (ATP) is a major metabolite that is essential for all living organisms. Therefore, a clearer understanding of ATP dynamics in living yeast cells is important for deciphering cellular energy ...
    A Protocol to Map the Spatial Proteome Using HyperLOPIT in Saccharomyces cerevisiae
    Authors:  Daniel J. H. Nightingale, Kathryn S. Lilley and Stephen G. Oliver, date: 07/20/2019, view: 1010, Q&A: 0
    [Abstract] The correct subcellular localization of proteins is vital for cellular function and the study of this process at the systems level will therefore enrich our understanding of the roles of proteins within the cell. Multiple methods are available for the study of protein subcellular localization, including fluorescence microscopy, organelle ...
    Preparation of RNA 3’ End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae
    Authors:  Manfred Schmid, Agnieszka Tudek and Torben Heick Jensen, date: 03/20/2019, view: 1408, Q&A: 0
    [Abstract] Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have ...
    Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure
    Authors:  Khoa Tran and Erin M. Green, date: 01/20/2019, view: 2956, Q&A: 0
    [Abstract] In the presence of oxidative stress, cellular defense systems that can detoxify reactive oxygen species are activated through multiple signaling cascades and transcriptional reprogramming. The budding yeast Saccharomyces cerevisiae has served as an excellent model for genetically-identifying factors important for the response to oxidative ...
    An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
    Authors:  Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 2051, Q&A: 0
    [Abstract] In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by ...
    Separation and Visualization of Low Abundant Ubiquitylated Forms
    Authors:  Ramona Schuster, Tânia Simões, Fabian den Brave and Mafalda Escobar-Henriques, date: 11/20/2018, view: 1604, Q&A: 0
    [Abstract] In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the ...
    Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
    Authors:  Congwei Wang, Julie Weidner and Anne Spang, date: 10/05/2018, view: 2519, Q&A: 0
    [Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, ...



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