Protocols in Current Issue
    In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 29, Q&A: 0
    [Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other ...
    Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts
    Authors:  Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 30, Q&A: 0
    [Abstract] Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the ...
    Protease Activity Assay in Fly Intestines
    Authors:  Marie-Paule Nawrot-Esposito, Rihab Loudhaief and Armel Gallet, date: 09/20/2017, view: 43, Q&A: 0
    [Abstract] The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the ...
    Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels
    Authors:  Marta Rodríguez-Ruiz, Paulo T. Mioto, José M. Palma and Francisco J. Corpas , date: 09/20/2017, view: 22, Q&A: 0
    [Abstract] In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems (King et al., 2005) for plant samples. Briefly, ...
    Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
    Authors:  Steven Boeynaems, Mathias De Decker, Peter Tompa and Ludo Van Den Bosch, date: 09/05/2017, view: 372, Q&A: 0
    [Abstract] Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems ...
    Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
    Authors:  Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 314, Q&A: 0
    [Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...
    Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells
    Authors:  Huy Nguyen Duc and Xiaojun Ren , date: 09/05/2017, view: 178, Q&A: 0
    [Abstract] HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
    Ubiquitin Proteasome Activity Measurement in Total Plant Extracts
    Authors:  Suayib Üstün and Frederik Börnke, date: 09/05/2017, view: 274, Q&A: 0
    [Abstract] The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to ...
    [Bio101] Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example
    Authors:  Yong Joon Kim, Stefan Kaluz, Anil Mehta, Emily Weinert, Shannon Rivera and Erwin G. Van Meir, date: 09/05/2017, view: 134, Q&A: 0
    [Abstract] The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, ...
    Assessment of Modulation of Protein Stability Using Pulse-chase Method
    Author:  Mohamed Elgendy, date: 08/20/2017, view: 403, Q&A: 0
    [Abstract] Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows ...