• Volume 11, 2021
  • Volume 10, 2020
  • Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011
Image Credit: BioProtoc.4127

Past Issues in 2021

Volume: 11 Issue: 16

Biochemistry

Monitoring Protein Splicing Using In-gel Fluorescence Immediately Following SDS-PAGE Authors:  Joel Weinberger II and Christopher W. Lennon, date: 08/20/2021, view: 1178, Q&A: 0

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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Oil Red O Staining for Lipid Content in Caenorhabditis elegans Authors:  Feng-Yung Wang and Tsui-Ting Ching, date: 08/20/2021, view: 893, Q&A: 0

The nematode Caenorhabditis elegans has emerged as a popular model system for studying the regulation of lipid metabolism. Therefore, it is critical to develop a method for determining fat storage in individual worms. Oil Red O (ORO) staining has been validated as an accurate assessment for major fat storage in C. elegans. Here, we describe an

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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: 1350, Q&A: 0

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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Biological Engineering

3D-printed Recoverable Microdrive and Base Plate System for Rodent Electrophysiology Authors:  Mihály Vöröslakos, Hiroyuki Miyawaki, Sebastien Royer, Kamran Diba, Euisik Yoon, Peter C. Petersen and György Buzsáki, date: 08/20/2021, view: 871, Q&A: 0

Extracellular recordings in freely moving animals allow the monitoring of brain activity from populations of neurons at single-spike temporal resolution. While state-of-the-art electrophysiological recording devices have been developed in recent years (e.g., µLED and Neuropixels silicon probes), implantation methods for silicon probes in

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Biophysics

Unraveling the Physicochemical Determinants of Protein Liquid-liquid Phase Separation by Nanoscale Infrared Vibrational Spectroscopy Authors:  Francesco S. Ruggeri, Alyssa M. Miller, Michele Vendruscolo and Tuomas P. J. Knowles, date: 08/20/2021, view: 919, Q&A: 0

The phenomenon of reversible liquid-liquid phase separation of proteins underlies the formation of membraneless organelles, which are crucial for cellular processes such as signalling and transport. In addition, it is also of great interest to uncover the mechanisms of further irreversible maturation of the functional dense liquid phase into

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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: 682, Q&A: 0

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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Cancer Biology

Measuring DNA Damage Using the Alkaline Comet Assay in Cultured Cells Authors:  Elena Clementi, Zuzana Garajova and Enni Markkanen, date: 08/20/2021, view: 966, Q&A: 0

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA-damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic

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Cell Biology

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: 956, Q&A: 0

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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Developmental Biology

Preparation of Drosophila Larval Blood Cells for Single-cell RNA Sequencing Authors:  Sudhir Gopal Tattikota and Norbert Perrimon, date: 08/20/2021, view: 537, Q&A: 0

Recent advances in single-cell RNA-sequencing (scRNA-seq) technologies provide unprecedented opportunities to identify new cell types and characterize cell states. One of the most important requirements for performing scRNA-seq is to obtain high-quality single cells in suspension. Recently, we used this approach to characterize Drosophila blood

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Immunology

Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies Authors:  Taishi Kondo, Minori Eguchi, Nariaki Tsuzuki, Naoya Murata, Tomoshige Fujino, Gosuke Hayashi and Hiroshi Murakami, date: 08/20/2021, view: 536, Q&A: 0

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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One-step White Blood Cell Extracellular Staining Method for Flow Cytometry Authors:  Ines Ait Belkacem, Pénélope Bourgoin, Jean Marc Busnel, Franck Galland and Fabrice Malergue, date: 08/20/2021, view: 688, Q&A: 0

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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Microbiology

Acid Hydrolysis for the Extraction of Archaeal Core Lipids and HPLC-MS Analysis Authors:  Maxime Tourte, Philippe Schaeffer, Vincent Grossi and Philippe M. Oger, date: 08/20/2021, view: 392, Q&A: 0

Lipid membranes are essential cellular elements as they provide cellular integrity and selective permeability under a broad range of environmental settings upon cell growth. In particular, Archaea are commonly recognized for their tolerance to extreme conditions, which is now widely accepted to stem from the unique structure of their lipids. While

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Development and Quantitation of Pseudomonas aeruginosa Biofilms after in vitro Cultivation in Flow-reactors Authors:  Yingdan Zhang, Jingru Zhao, Hang Cheng, Jing Wang, Liang Yang and Haihua Liang, date: 08/20/2021, view: 383, Q&A: 0

Characterization of biofilm formation and metabolic activities is critical to investigating biofilm interactions with environmental factors and illustrating biofilm regulatory mechanisms. An appropriate in vitro model that mimics biofilm in vivo habitats therefore demands accurate quantitation and investigation of biofilm-associated activities.

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Molecular Biology

Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike Authors:  Leila Mekkaoui, Emma M. Bentley, Mathieu Ferrari, Katarina Lamb, Katarzyna Ward, Rajeev Karattil, Zulaikha Akbar, Reyisa Bughda, James Sillibourne, Shimobi Onuoha, Giada Mattiuzzo, Yasu Takeuchi and Martin Pule, date: 08/20/2021, view: 408, Q&A: 0

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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Plant Science

A Novel Method to Map Small RNAs with High Resolution Authors:  Kun Huang, Feray Demirci, Blake C. Meyers and Jeffrey L. Caplan, date: 08/20/2021, view: 620, Q&A: 0

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect

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Tomato Stem Injection for the Precise Assessment of Ralstonia solanacearum Fitness in Planta Authors:  Yaru Wang, Liu Xian, Gang Yu and Alberto P. Macho, date: 08/20/2021, view: 460, Q&A: 0

Ralstonia solanacearum is a soil-borne pathogen with worldwide distribution that causes bacterial wilt disease in more than 250 plant species. R. solanacearum invades plants through the roots, reaches the vascular system, and colonizes the whole plant by moving through the xylem, where it eventually replicates rapidly, causing plant death. Usual

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Stem Cell

Isolation of Single Cells from Mouse Periodontal Ligament Authors:  Jing Zhao and Paul Sharpe, date: 08/20/2021, view: 897, Q&A: 0

The periodontal ligament (PDL) is an essential tissue connecting teeth and bone. It is a complex tissue specifically designed to absorb the forces of mastication; analysis of its multiple cell populations is important to understand its function and the cell changes associated with periodontal disease. Cells in the periodontal ligament are not

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Neutral Comet Assay to Detect and Quantitate DNA Double-Strand Breaks in Hematopoietic Stem Cells Authors:  Irene Mariam Roy, Pon Sowbhagya Nadar and Satish Khurana, date: 08/20/2021, view: 526, Q&A: 0

In vertebrates, hematopoietic stem cells (HSCs) regulate the supply of blood cells throughout the lifetime and help to maintain homeostasis. Due to their long lifespan, genetic integrity is paramount for these cells, and accordingly, a number of stem cell-specific mechanisms are employed. However, HSCs tend to show more DNA damage with increasing

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