Protocols in Current Issue
    A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro
    Authors:  Andreas Holleufer and Rune Hartmann, date: 10/20/2018, view: 1293, Q&A: 0
    [Abstract] Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate ...
    Immunogold Electron Microscopy of the Autophagosome Marker LC3
    Authors:  Domenico Mattoscio, Andrea Raimondi, Carlo Tacchetti and Susanna Chiocca, date: 12/20/2017, view: 4871, Q&A: 0
    [Abstract] Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents ...
    Detection of ASC Oligomerization by Western Blotting
    Authors:  Jérôme Lugrin and Fabio Martinon , date: 05/20/2017, view: 8047, Q&A: 0
    [Abstract] The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote ...
    Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
    Authors:  Noelia Lander, Miguel A. Chiurillo, Aníbal E. Vercesi and Roberto Docampo, date: 05/20/2017, view: 7417, Q&A: 0
    [Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then ...
    Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
    Authors:  Christin Herrmann, Daphne C. Avgousti and Matthew D. Weitzman, date: 03/20/2017, view: 8049, Q&A: 0
    [Abstract] Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome ...
    Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents
    [Abstract] The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). ...
    Isolation of Particles of Recombinant ASC and NLRP3
    Authors:  Fátima Martín-Sánchez, Ana I. Gómez and Pablo Pelegrín, date: 05/20/2015, view: 7319, Q&A: 0
    [Abstract] NLRP3 inflammasome is a multiprotein complex responsible for the activation of inflammatory caspase-1, resulting in processing and release of pro-inflammatory cytoquines IL-1β and IL-18 (Schroder and Tschopp, 2010). This inflammasome is composed of the sensor protein NLRP3 connected to caspase-1 through the adaptor protein ASC ...
    Chromogenic Substrate Assay for Determining the Activity of Plasma Kallikrein
    Authors:  Praveen Papareddy, Martina Kalle and Artur Schmidtchen, date: 10/05/2014, view: 5926, Q&A: 0
    [Abstract] The activation of the intrinsic pathway takes place at negatively charged surfaces, such as bacteria, and involves activation of cogulation Factor XII, which then leads to the activation of plasma kallikrein (PK) and coagulation Factor XI. To determine the PK activity on bacterial surfaces, bacteria were pre-incubated with peptides, followed by ...
    Coagulation Assay
    Authors:  Praveen Papareddy, Martina Kalle and Artur Schmidtchen, date: 10/05/2014, view: 8609, Q&A: 0
    [Abstract] Clotting times can be measured by using citrate plasma. The intrinsic pathway of coagulation is measured by the activated partial thromboplastin time (aPTT), the extrinsic pathway of coagulation, monitored by measuring the prothrombin time (PT), and thrombin-induced fibrin-network formation (thrombin clotting time; TCT).
    Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages
    Authors:  Sargurunathan Subashchandrabose and Harry L.T. Mobley, date: 09/20/2014, view: 15366, Q&A: 0
    [Abstract] Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 ...

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