Protocols in Current Issue
    Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure
    Authors:  Khoa Tran and Erin M. Green, date: 01/20/2019, view: 1815, 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 ...
    Functional Evaluation of the Signal Peptides of Secreted Proteins
    Authors:  Weixiao Yin, Yufu Wang, Tao Chen, Yang Lin and Chaoxi Luo, date: 05/05/2018, view: 2507, Q&A: 2
    [Abstract] Here, we describe a method that can be used to evaluate the function of predicted signal peptides. This method utilizes the yeast Saccharomyces cerevisiae YTK12 strain and pSUC2 vector in which the pSUC2 vector with fused predicted signal peptide is transformed into yeast. The function of the signal peptides can be evaluated by using ...
    Metal-tagging Transmission Electron Microscopy for Localisation of Tombusvirus Replication Compartments in Yeast
    Authors:  Isabel Fernández de Castro and Cristina Risco, date: 04/20/2018, view: 2068, Q&A: 0
    [Abstract] Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication ...
    Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae
    Authors:  Sviatlana Shashkova, Adam JM Wollman, Stefan Hohmann and Mark C Leake, date: 01/20/2018, view: 3082, Q&A: 0
    [Abstract] Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule ...
    A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae
    Authors:  Vidhya Bharathi, Amandeep Girdhar and Basant K Patel, date: 08/05/2017, view: 4612, Q&A: 0
    [Abstract] The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are ...
    Uptake Assay for Radiolabeled Peptides in Yeast
    Authors:  Melinda Hauser, Houjian Cai, Fred Naider and Jeffrey M. Becker, date: 11/20/2016, view: 4501, Q&A: 0
    [Abstract] We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.
    Halo Assay for Toxic Peptides and Other Compounds in Microorganisms
    Authors:  Houjian Cai, Melinda Hauser, Fred Naider and Jeffrey M. Becker, date: 11/20/2016, view: 4696, Q&A: 0
    [Abstract] We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of ...
    Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures
    Authors:  Cheng-Hsun Ho and Wolf B. Frommer, date: 02/05/2016, view: 5584, Q&A: 0
    [Abstract] This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of ...
    Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors
    Authors:  Cindy Ast, Wolf B. Frommer, Guido Grossmann and Roberto De Michele, date: 01/05/2015, view: 7000, Q&A: 0
    [Abstract] AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two ...
    Synchronization of Saccharomyces cerevisiae Cells in G1 Phase of the Cell Cycle
    Authors:  Ildiko Unk and Andreea Daraba, date: 10/20/2014, view: 10679, Q&A: 0
    [Abstract] The baker’s yeast, Saccharomyces cerevisiae is a widely used model organism in molecular biology because of the high homology it shares with human cells in many basic cellular processes such as DNA replication, repair, recombination, transcription, and because of the ease its genome can be manipulated. Other advantages of working with ...

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