Molecular Biology

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    Protocols in Current Issue
    In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
    Authors:  Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 831, Q&A: 0
    [Abstract] It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the ...
    Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages
    Authors:  Moumita Basu, Shriya Saha and Anindita Ukil, date: 04/05/2020, view: 619, Q&A: 0
    [Abstract] Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different ...
    Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method
    Authors:  Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 758, Q&A: 0
    [Abstract] This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (Em = -55 mV) in the SdhA flavoprotein subunit of succinate ...
    Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay
    Authors:  Connor M. Blair, Jiayue Ling and George S. Baillie, date: 04/05/2020, view: 485, Q&A: 0
    [Abstract] Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE ...
    Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
    Authors:  Louise Thines and Pierre Morsomme, date: 03/05/2020, view: 834, Q&A: 0
    [Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature ...
    A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s)
    Authors:  Antonia Efstathiou and Despina Smirlis, date: 01/20/2020, view: 645, Q&A: 0
    [Abstract] The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s ...
    In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
    Authors:  G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 2548, Q&A: 0
    [Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...
    In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria
    Authors:  Krishna K. Singh, Devendra P. Singh, Rambir Singh and Deepak K. Saini, date: 07/05/2019, view: 1728, Q&A: 0
    [Abstract] Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic ...
    Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli
    Authors:  Nanami Kawaguchi, Haruko Ogawa and Kimie Date, date: 07/05/2019, view: 1613, Q&A: 0
    [Abstract] Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from ...
    Transient Expression Assay in Strawberry Fruits
    Authors:  Mengting Pi, Qi Gao and Chunying Kang, date: 06/05/2019, view: 2581, Q&A: 0
    [Abstract] Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, ...



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