Biochemistry

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    The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein
    Authors:  Amita Kaundal, Vemanna S. Ramu and Kirankumar S. Mysore, date: 08/05/2020, view: 92, Q&A: 0
    [Abstract] ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In ...
    A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
    Authors:  Paul J. Steiner, Zachary T Baumer and Timothy A. Whitehead, date: 08/05/2020, view: 126, Q&A: 0
    [Abstract] Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking ...
    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 ...
    In vitro Induction and Detection of Acrosomal Exocytosis in Human Spermatozoa
    Authors:  Shenae L. Cafe, Amanda L. Anderson and Brett Nixon, date: 07/20/2020, view: 253, Q&A: 0
    [Abstract] The acrosome reaction is a highly regulated exocytotic event that primes spermatozoa for successful fertilization. Upon induction, acrosomal exocytosis proceeds via a wave of vesiculation that radiates across the sperm head, destabilizing the acrosomal vesicle and resulting in the release of the acrosomal contents. Having shed their acrosome, ...
    Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
    Authors:  Shaista Anwer and Katalin Szaszi, date: 07/20/2020, view: 328, Q&A: 0
    [Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows ...
    Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
    Authors:  Johanna M. Schafer and Jennifer A. Pietenpol, date: 07/05/2020, view: 565, Q&A: 0
    [Abstract] MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system ...
    Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
    Authors:  Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 584, Q&A: 0
    [Abstract] Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...
    In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance
    [Abstract] The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...
    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: 827, 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 ...



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