Protocols in Current Issue
    Protocol for Peptide Synthesis on Spectrally Encoded Beads for MRBLE-pep Assays
    Authors:  Jamin B. Hein, Huy Q. Nguyen, Martha Cyert and Polly M. Fordyce, date: 07/05/2020, view: 41, Q&A: 0
    [Abstract] Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs). In many cases, these PPIs are between a folded protein domain and a short linear motif (SLiM) within an unstructured region of a protein. As a result of this small interaction interface (3-10 amino acids), the affinities of SLiM-mediated ...
    Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
    [Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...
    In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
    Authors:  Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 754, 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 ...
    Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease
    Authors:  Yibing Deng, Shengchao Shi, Bin Zheng, Tao Wu and Peng Zheng, date: 04/20/2020, view: 984, Q&A: 0
    [Abstract] The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the ...
    An Optimized Method for the Production Using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes
    Authors:  George Carnell, Keith Grehan, Francesca Ferrara, Eleonora Molesti and Nigel Temperton, date: 08/20/2017, view: 8491, Q&A: 0
    [Abstract] The protocol outlined represents a cost-effective, rapid and reliable method for the generation of high-titre viral pseudotype particles with the wild-type SARS-CoV spike protein on a lentiviral vector core using the widely available transfection reagent PEI. This protocol is optimized for transfection in 6-well plates; however it can be readily ...
    Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)
    Authors:  Nikos Kourtis and Nektarios Tavernarakis, date: 03/05/2017, view: 5947, Q&A: 0
    [Abstract] Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.
    Analysis of Protein Stability by the Cycloheximide Chase Assay
    [Abstract] Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a ...
    Development of a Novel Assay for Synthesis and Hydrolysis of Sedoheptulose 1,7-bisphosphate (SBP) in vitro by Combinations of Purified Fructose 1,6-bisphosphate aldolases (FBA) Proteins and Fructose 1,6-bisphosphatases (FBPase) Proteins from Bacillus methanolicus MGA3
    [Abstract] Bacillus methanolicus (B. methanolicus) is a Gram-positive, thermotolerant, and facultative methylotrophic bacterium that can use the one-carbon (C1) compound methanol as a source of carbon and energy (Schendel et al., 1990; Arfman et al., 1997; Arfman et al., 1992). B. methanolicus uses the ...
    Peptide Synthesis
    Authors:  Zheng Miao and Zhen Cheng, date: 07/20/2012, view: 22618, Q&A: 2
    [Abstract] This protocol describes the synthesis of peptides for affinity testing and bioconjugate with solid phase peptide synthesizer at a small scale.

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