Protocols in Current Issue
    Amber Suppression Technology for Mapping Site-specific Viral-host Protein Interactions in Mammalian Cells
    Authors:  Nur Firdaus Isa, Olivier Bensaude and Shona Murphy, date: 02/05/2022, view: 1124, Q&A: 0

    Probing the molecular interactions of viral-host protein complexes to understand pathogenicity is essential in modern virology to help the development of antiviral therapies. Common binding assays, such as co-immunoprecipitation or pull-downs, are helpful in investigating intricate viral-host proteins interactions. However, such assays may miss

    Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

    Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long

    Fe-NTA Microcolumn Purification of Phosphopeptides from Immunoprecipitation (IP) Eluates for Mass Spectrometry Analysis
    Authors:  Ethan J. Sanford and Marcus B. Smolka, date: 08/05/2021, view: 1495, Q&A: 0

    Protein phosphorylation is a nearly universal signaling mechanism. To date, a number of proteomics tools have been developed to analyze phosphorylation. Phosphoproteome-wide analyses using whole cell extracts suffer from incomplete coverage, often missing phosphorylation events from low-abundance proteins. In order to increase coverage of

    Expression and Purification of the Human Cation-chloride Cotransporter KCC1 from HEK293F Cells for Structural Studies
    Authors:  Si Liu and Jiangtao Guo, date: 04/05/2021, view: 1574, Q&A: 0

    Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations such as Na+ and/or K+ with chloride across membrane. Among CCCs family, K-Cl cotransporters (KCC1-KCC4) extrude intracellular Cl- by the transmembrane K+ gradient. In humans, these KCCs play vital roles in the physiology of the nervous system and kidney.

    In vitro Measurement of Membrane Attack Complex in RPE Cells
    Authors:  Kelly Mulfaul and Sarah L. Doyle, date: 02/20/2021, view: 2478, Q&A: 0

    Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the

    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 3453, Q&A: 0

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of

    In vitro Cultivation and Visualization of Malaria Liver Stages in Primary Simian Hepatocytes
    Authors:  Devendra Kumar Gupta and Thierry Diagana, date: 08/20/2020, view: 2822, Q&A: 0
    [Abstract] Human liver is the primary and obligatory site for malaria infection where sporozoites invade host hepatocytes. Malaria hepatic stages are asymptomatic and represent an attractive target for development of anti-malarial interventions and vaccines. However, owing to lack of robust and reproducible in vitro culture system, it is difficult ...
    Production and Isolation of Magnetic Protein Crystals in HEK293T Cells
    Authors:  Thomas L. Li and Bianxiao Cui, date: 07/20/2020, view: 2510, 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 ...
    Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins
    [Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...
    Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration
    Authors:  Joni Vanneste, Thomas Vercruysse, Philip Van Damme, Ludo Van Den Bosch and Dirk Daelemans, date: 06/20/2020, view: 3118, Q&A: 0
    [Abstract] Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to ...

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