• 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.4100

Current Issue in 2021

Volume: 11 Issue: 14

Biochemistry

A Gel-Based Assay for Probing Protein Translocation on dsDNA Authors:  Christiane Brugger and Alexandra M. Deaconescu, date: 07/20/2021, view: 89, Q&A: 0

Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are

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Protocol for Spontaneous and Chaperonin-assisted in vitro Refolding of a Slow-folding Mutant of GFP, sGFP Authors:  Anwar Sadat, Satyam Tiwari and Koyeli Mapa, date: 07/20/2021, view: 85, Q&A: 0

Understanding the folding pathway of any protein is of utmost importance for deciphering the folding problems under adverse conditions. We can obtain important information about the folding pathway by monitoring the folding of any protein from its unfolded state. It is usually very difficult to monitor the folding process in real time as the

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Biophysics

A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 75, Q&A: 0

Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind

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Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 186, Q&A: 0

Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

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Cancer Biology

Modeling the Nonlinear Dynamics of Intracellular Signaling Networks Authors:  Oleksii S. Rukhlenko and Boris N. Kholodenko, date: 07/20/2021, view: 116, Q&A: 0

This protocol illustrates a pipeline for modeling the nonlinear behavior of intracellular signaling pathways. At fixed spatial points, nonlinear signaling dynamics are described by ordinary differential equations (ODEs). At constant parameters, these ODEs may have multiple attractors, such as multiple steady states or limit cycles. Standard

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Cell Biology

Preparation of Human Chondrocytes for Profiling Using Cytometry by Time-of-flight (cyTOF) Authors:  Fiorella Carla Grandi and Nidhi Bhutani, date: 07/20/2021, view: 84, Q&A: 0

Single-cell technologies have allowed high-resolution profiling of tissues and thus a deeper understanding of tissue homeostasis and disease heterogeneity. Understanding this heterogeneity can be especially important for tailoring treatments in a patient-specific manner. Here, we detail methods for preparing human cartilage tissue for profiling

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Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep Authors:  Francesco Ciscato, Federica Chiara, Riccardo Filadi and Andrea Rasola, date: 07/20/2021, view: 109, Q&A: 0

The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective

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Developmental Biology

GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish Authors:  Jordan M. Welker, Wesley A. Wierson, Maira P. Almeida, Carla M. Mann, Melanie E. Torrie, Zhitao Ming, Stephen C. Ekker, Karl J. Clark, Drena L. Dobbs, Jeffrey J. Essner and Maura McGrail, date: 07/20/2021, view: 114, Q&A: 0

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging)

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Immunology

Isolation of Microglia and Analysis of Protein Expression by Flow Cytometry: Avoiding the Pitfall of Microglia Background Autofluorescence Authors:  Jeremy C. Burns, Richard M. Ransohoff and Michaël Mingueneau, date: 07/20/2021, view: 104, Q&A: 0

Microglia are a unique type of tissue-resident innate immune cell found within the brain, spinal cord, and retina. In the healthy nervous system, their main functions are to defend the tissue against infectious microbes, support neuronal networks through synapse remodeling, and clear extracellular debris and dying cells through phagocytosis. Many

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Microbiology

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract Authors:  Wei Tan, Zhi-Hui Shao and Guo-Ping Zhao, date: 07/20/2021, view: 78, Q&A: 0

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate

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Molecular Biology

Antisense Oligo Pulldown of Circular RNA for Downstream Analysis Authors:  Debojyoti Das, Aniruddha Das and Amaresh C. Panda, date: 07/20/2021, view: 114, Q&A: 0

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect

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A Simple Method to Generate Super-sensitive AID (ssAID)-based Conditional Knockouts using CRISPR-based Gene Knockout in Various Vertebrate Cell Lines Authors:  Kohei Nishimura and Tatsuo Fukagawa, date: 07/20/2021, view: 86, Q&A: 0

Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to

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Neuroscience

Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts Authors:  Corey R. Landry, Mighten C. Yip, Ilya Kolb, William A. Stoy, Mercedes M. Gonzalez and Craig R. Forest, date: 07/20/2021, view: 211, Q&A: 0

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This

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Plant Science

Spatiotemporal Quantification of Cytosolic pH in Arabidopsis Pollen Tubes Authors:  Maria Teresa Portes, Daniel S.C. Damineli and José A. Feijó, date: 07/20/2021, view: 154, Q&A: 0

Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports

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