Protocols in Current Issue
    Systematic Quantification of GFP-tagged Protein Foci in Schizosaccharomyces pombe Nuclei
    Authors:  Kim Kiat Lim and Ee Sin Chen, date: 12/20/2018, view: 205, Q&A: 0
    [Abstract] DNA damage repair proteins form foci in response to DNA damaging agents. The efficiency and integrity of the DNA repair pathway of a particular eukaryotic (mutant) strain is usually determined by the number of foci formed compared with their wild-type counterpart. Conventionally, focus number is determined visually, and this low accuracy may ...
    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: 396, 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 ...
    Field Inoculation and Classification of Maize Ear Rot Caused by Fusarium verticillioides
    Authors:  Chaopei Dong, Yabin Wu, Jingyang Gao, Zijian Zhou, Cong Mu, Peipei Ma, Jiafa Chen and Jianyu Wu, date: 12/05/2018, view: 233, Q&A: 0
    [Abstract] Maize ear rot is a worldwide fungal disease mainly caused by Fusarium verticillioides and Fusarium graminearum. Maize planted in the field was inoculated with Fusarium verticillioides at the filling stage, 15 days after pollination. Two milliliters of spore suspension with a concentration of 5 x 106/ml was ...
    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: 575, 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 and Purification of Proteins Secreted from Phytophthora sojae
    Authors:  Yeqiang Xia, Yan Wang and Yuanchao Wang, date: 10/20/2018, view: 644, Q&A: 0
    [Abstract] Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and ...
    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: 754, 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, ...
    Identifying Protein Interactions with Histone Peptides Using Bio-layer Interferometry
    Authors:  Bingbing Ren, Ahmed Mahmoud Mohammed Sayed, Hwei Ling Tan, Yu Keung Mok and Ee Sin Chen, date: 09/20/2018, view: 771, Q&A: 0
    [Abstract] Histone post-translational modifications (PTMs) regulate numerous cellular processes, including gene transcription, cell division, and DNA damage repair. Most histone PTMs affect the recruitment or exclusion of reader proteins from chromatin. Here, we present a protocol to measure affinity and interaction kinetics between histone peptides and the ...
    Extracting and Integrating Protein Localization Changes from Multiple Image Screens of Yeast Cells
    Authors:  Alex X Lu, Louis-Francois Handfield and Alan M Moses, date: 09/20/2018, view: 564, Q&A: 0
    [Abstract] The evaluation of protein localization changes in cells under diverse chemical and genetic perturbations is now possible due to the increasing quantity of screens that systematically image thousands of proteins in an organism. Integrating information from different screens provides valuable contextual information about the protein function. For ...
    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: 591, 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 ...
    Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth
    Authors:  Yvonne Becker, Kimberly A. Green, Barry Scott and Matthias Becker, date: 09/05/2018, view: 620, Q&A: 0
    [Abstract] Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic ...

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