Protocols in Current Issue
    Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method
    Authors:  Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 310, Q&A: 0
    [Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...
    Enzymatic Assays and Enzyme Histochemistry of Tuta absoluta Feeding on Tomato Leaves
    Authors:  Rim Hamza, José P. Beltrán and Luis A. Cañas, date: 09/05/2018, view: 286, Q&A: 0
    [Abstract] Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed ...
    Measuring CD38 Hydrolase and Cyclase Activities: 1,N6-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) Fluorescence-based Methods
    [Abstract] CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD+) metabolism. Through its major activity, the hydrolysis of NAD+, CD38 helps maintain the appropriate levels of this molecule for all NAD+-dependent metabolic processes to occur. Due to current advances and ...
    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: 811, 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: 849, 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 ...
    Enzymatic Activity Assay for Invertase in Synechocystis Cells
    Authors:  Xiaoming Tan, Kuo Song and Xuefeng Lu, date: 05/20/2018, view: 775, Q&A: 0
    [Abstract] Invertase can catalyze the hydrolysis of sucrose, and is widely distributed in cells of cyanobacteria and plants. Being responsible for the first step for sucrose metabolism, invertase plays important physiological roles and its enzymatic activity is frequently needed to be determined. All the methods for determination of the invertase activity ...
    In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts
    Authors:  Joo Yong Kim and Hak Soo Seo, date: 04/05/2018, view: 1287, Q&A: 0
    [Abstract] Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude ...
    Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP
    Authors:  Tomoharu Kanie and Peter K. Jackson, date: 04/05/2018, view: 1863, Q&A: 0
    [Abstract] GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct ...
    Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
    Authors:  Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 1225, Q&A: 0
    [Abstract] Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack ...
    Infectious Subviral Particle-induced Hemolysis Assay for Mammalian Orthoreovirus
    Authors:  Anthony J. Snyder and Pranav Danthi, date: 01/20/2018, view: 911, Q&A: 0
    [Abstract] Mammalian orthoreovirus (reovirus) utilizes pore forming peptides to penetrate host cell membranes. This step is essential for delivering its genome containing core particle during viral entry. This protocol describes an in vitro assay for measuring reovirus-induced pore formation.