Protocols in Current Issue
    Isolation of Thylakoid Membranes from the Cyanobacterium Synechocystis sp. PCC 6803 and Analysis of Their Photosynthetic Pigment-protein Complexes by Clear Native-PAGE
    Authors:  Josef Komenda, Vendula Krynická and Tomas Zakar, date: 01/05/2019, view: 1046, Q&A: 0
    [Abstract] Cyanobacteria represent a frequently used model organism for the study of oxygenic photosynthesis. They belong to prokaryotic microorganisms but their photosynthetic apparatus is quite similar to that found in algal and plant chloroplasts. The key players in light reactions of photosynthesis are Photosystem I and Photosystem II complexes (PSI and ...
    Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP
    Authors:  Heather B. Case and Nicholas E. Dickenson, date: 11/05/2018, view: 993, Q&A: 0
    [Abstract] ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase ...
    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: 1597, 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 ...
    Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
    Authors:  Tyler Milston Renner, Kasandra Bélanger and Marc-André Langlois, date: 09/05/2018, view: 1234, Q&A: 0
    [Abstract] There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly ...
    A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae
    Authors:  Jyumpei Kobayashi, Daisuke Sasaki and Akihiko Kondo, date: 06/20/2018, view: 2181, Q&A: 0
    [Abstract] In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell ...
    Characterization of Protein Domain Function via in vitro DNA Shuffling
    Authors:  Kathy Hiu Laam Po, Edward Wai Chi Chan and Sheng Chen, date: 06/05/2018, view: 1506, Q&A: 0
    [Abstract] We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active ...
    Detection of Catalase Activity by Polyacrylamide Gel Electrophoresis (PAGE) in Cell Extracts from Pseudomonas aeruginosa
    Authors:  Magdalena Pezzoni, Ramón A. Pizarro and Cristina S. Costa, date: 06/05/2018, view: 2092, Q&A: 0
    [Abstract] Bacteria in nature and as pathogens commonly face oxidative stress which causes damage to proteins, lipids and DNA. This damage is produced by the action of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), singlet oxygen, superoxide anion and hydroxyl radical. ROS are generated by antimicrobials, environmental ...
    In vitro Analysis of Ubiquitin-like Protein Modification in Archaea
    Authors:  Xian Fu, Zachary Adams and Julie A. Maupin-Furlow, date: 05/20/2018, view: 1817, Q&A: 0
    [Abstract] The ubiquitin-like (Ubl) protein is widely distributed in Archaea and involved in many cellular pathways. A well-established method to reconstitute archaeal Ubl protein conjugation in vitro is important to better understand the process of archaeal Ubl protein modification. This protocol describes the in vitro reconstitution of ...
    Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
    Authors:  Prarthana Mohanraju, John van der Oost, Martin Jinek and Daan C. Swarts, date: 05/05/2018, view: 3942, Q&A: 3
    [Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for ...
    Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
    Authors:  Kuancheng Liu and Jianming Hu, date: 04/20/2018, view: 1997, Q&A: 0
    [Abstract] The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for ...

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