Protocols in Current Issue
    Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis
    Authors:  Sujeet Kumar and Natividad Ruiz , date: 06/20/2019, view: 154, Q&A: 0
    [Abstract] Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of ...
    Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
    Authors:  Joseph McInnes, Lujia Zhou and Patrik Verstreken, date: 11/20/2018, view: 1459, Q&A: 0
    [Abstract] Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the ...
    Microtitre Plate Based Cell-SELEX Method
    Authors:  Munish Shorie and Harmanjit Kaur, date: 10/20/2018, view: 1583, Q&A: 0
    [Abstract] Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. We report a facile microtiter plate-based ...
    A Method for SUMO Modification of Proteins in vitro
    Authors:  Christine C. Lee, Bing Li, Hongtao Yu and Michael J. Matunis, date: 10/05/2018, view: 1478, Q&A: 0
    [Abstract] The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin ...
    Bacterial Microcolonies in Gel Beads for High-throughput Screening
    Author:  Yolanda Schaerli, date: 07/05/2018, view: 2809, Q&A: 0
    [Abstract] High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants.
    Implementation of Blue Light Switchable Bacterial Adhesion for Design of Biofilms
    Authors:  Fei Chen and Seraphine V. Wegner, date: 06/20/2018, view: 1974, Q&A: 0
    [Abstract] Control of bacterial adhesions to a substrate with high precision in space and time is important to form a well-defined biofilm. Here, we present a method to engineer bacteria such that they adhere specifically to substrates under blue light through the photoswitchable proteins nMag and pMag. This provides exquisite spatiotemporal remote control ...
    Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies
    Authors:  Mayuri Sadoine, Michele Cerminara, Jörg Fitter and Alexandros Katranidis, date: 06/20/2018, view: 2041, Q&A: 0
    [Abstract] Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET ...
    Characterization of Protein Domain Function via in vitro DNA Shuffling
    Authors:  Kathy Hiu Laam Po, Edward Wai Chi Chan and Sheng Chen, date: 06/05/2018, view: 1572, Q&A: 0
    [Abstract] We recently investigated the molecular events that drive evolution of the CTX-M-type β-lactamases by DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet the enzymatically active ...
    Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
    Authors:  Prarthana Mohanraju, John van der Oost, Martin Jinek and Daan C. Swarts, date: 05/05/2018, view: 4233, Q&A: 3
    [Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for ...
    Laminarin Quantification in Microalgae with Enzymes from Marine Microbes
    Authors:  Stefan Becker and Jan-Hendrik Hehemann, date: 04/20/2018, view: 2233, Q&A: 0
    [Abstract] The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy ...

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