Biochemistry

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    Protocols in Current Issue
    Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
    [Abstract] The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell ...
    Production and Isolation of Magnetic Protein Crystals in HEK293T Cells
    Authors:  Thomas L. Li and Bianxiao Cui, date: 07/20/2020, view: 425, Q&A: 0
    [Abstract] Advances in protein engineering have enabled the production of self-assembled protein crystals within living cells. Our recent publication demonstrates the production of ftn-PAK4, which is a ferritin-containing crystal that can mineralize iron and become magnetic when isolated. We have developed an optimized protocol for the production and ...
    Mouse Adipose Tissue Protein Extraction
    Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 887, Q&A: 0
    [Abstract] As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...
    Differential Fractionation of Erythrocytes Infected by Plasmodium berghei
    Authors:  Bénédicte Gnangnon, Véronique Peucelle and Christine Pierrot, date: 06/05/2020, view: 501, Q&A: 0
    [Abstract] The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected ...
    Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation
    Authors:  Abderrahim Benmoussa, Sara Michel, Caroline Gilbert and Patrick Provost, date: 06/05/2020, view: 1044, Q&A: 0
    [Abstract] Milk is a complex fluid that contains various types of proteins and extracellular vesicles (EVs). Some proteins can mingle with EVs, and interfere with their isolation. Among these proteins, caseins form micelles of a size comparable to milk EVs, and can thus be co-isolated with EVs. Preliminary steps that affect milk are crucial for EV isolation ...
    Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography
    Authors:  Minna M. Koskela, Petra Skotnicová, Éva Kiss and Roman Sobotka, date: 05/20/2020, view: 940, Q&A: 0
    [Abstract] Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model ...
    Isolation and Characterization of Exosomes from Mouse Feces
    Authors:  Chunhua Yang, Mingzhen Zhang, Junsik Sung, Lixin Wang, Yunjin Jung and Didier Merlin, date: 04/20/2020, view: 1213, Q&A: 0
    [Abstract] Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, ...
    Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins
    Authors:  Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 1003, Q&A: 0
    [Abstract] High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol ...
    Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System
    [Abstract] T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand ...
    Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry
    [Abstract] Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several ...



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