Biochemistry

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    Protocols in Current Issue
    Solubilization Method for Isolation of Photosynthetic Mega- and Super-complexes from Conifer Thylakoids
    Authors:  Pushan Bag, Wolfgang P. Schröder, Stefan Jansson and Domenica Farci, date: 09/05/2021, view: 445, Q&A: 0
    [Abstract]

    Photosynthesis is the main process by which sunlight is harvested and converted into chemical energy and has been a focal point of fundamental research in plant biology for decades. In higher plants, the process takes place in the thylakoid membranes where the two photosystems (PSI and PSII) are located. In the past few decades, the evolution of

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    Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike
    [Abstract]

    The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike

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    Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE
    Authors:  Joel Weinberger II and Christopher W. Lennon, date: 08/20/2021, view: 1123, Q&A: 0
    [Abstract]

    Inteins garner significant interest from both basic and applied researchers due to their unique catalytic abilities. Herein, we describe a protocol for accurately monitoring protein splicing without purification using in-gel fluorescence immediately following Tris-Glycine SDS-PAGE. Following expression in Escherichia coli, cells are lysed by

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    Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies
    [Abstract]

    Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody

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    Synchronized Real-time Measurement of Sec-mediated Protein Translocation
    Authors:  Riti Gupta, Dmitri Toptygin and Christian M. Kaiser, date: 08/20/2021, view: 617, Q&A: 0
    [Abstract]

    The Sec translocon, consisting of a heterotrimeric transmembrane channel (SecYEG) and an associated ATPase (SecA), catalyzes the export of unfolded proteins from the cytosol in bacteria. Kinetically resolving protein translocation at high resolution yields mechanistic insight into the process. Translocation is typically followed by measuring the

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    A Novel Method to Make Polyacrylamide Gels with Mechanical Properties Resembling those of Biological Tissues
    Authors:  Katarzyna Pogoda, Elisabeth E. Charrier and Paul A. Janmey, date: 08/20/2021, view: 897, Q&A: 0
    [Abstract]

    Studies characterizing how cells respond to the mechanical properties of their environment have been enabled by the use of soft elastomers and hydrogels as substrates for cell culture. A limitation of most such substrates is that, although their elastic properties can be accurately controlled, their viscous properties cannot, and cells

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    Implementing Novel Designs in pET Expression Plasmids that Increase Protein Production
    Authors:  Patrick J. Shilling and Daniel O. Daley, date: 08/20/2021, view: 1281, Q&A: 0
    [Abstract]

    pET expression plasmids are widely used in the biotechnology, biopharmaceutical, and basic research sectors for the production of recombinant proteins. Typically, they are used off-the-shelf because they support high production titers; however, we have identified two design flaws in many pET plasmids that limit their production capacity. We used

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    One-step White Blood Cell Extracellular Staining Method for Flow Cytometry
    [Abstract]

    Flow cytometry is a powerful analytical technique that is increasingly used in scientific investigations and healthcare; however, it requires time-consuming, multi-step sample procedures, which limits its use to specialized laboratories. In this study, we propose a new universal one-step method in which white blood cell staining and red blood cell

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

    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

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    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 960, Q&A: 0
    [Abstract]

    Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells

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