• 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.4017

Past Issues in 2021

Volume: 11 Issue: 10

Biochemistry

Cell-free Synthesis of Correctly Folded Proteins with Multiple Disulphide Bonds: Production of Fungal Hydrophobins Authors:  Rezwan Siddiquee and Ann H Kwan, date: 05/20/2021, view: 1144, Q&A: 0

Cell-free synthesis is a powerful technique that uses the transcriptional and translational machinery extracted from cells to create proteins without the constraints of living cells. Here, we report a cell-free protein production protocol using Escherichia coli lysate (Figure 1) to successfully express a class of proteins (known as hydrophobins)

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Intracellular IRF5 Dimerization Assay Authors:  Cherrie D. Sherman and Betsy J. Barnes, date: 05/20/2021, view: 821, Q&A: 0

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5

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A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins Authors:  Javier Aguilera, Salvador Vazquez-Reyes and Jianjun Sun, date: 05/20/2021, view: 787, Q&A: 0

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have

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Biophysics

Single-Molecule Studies of Membrane Receptors from Brain Region Specific Nanovesicles Authors:  Surya P. Aryal, Xu Fu, Abdullah A. Masud, Khaga R. Neupane and Christopher I. Richards, date: 05/20/2021, view: 728, Q&A: 0

Single molecule imaging and spectroscopy are powerful techniques for the study of a wide range of biological processes including protein assembly and trafficking. However, in vivo single molecule imaging of biomolecules has been challenging because of difficulties associated with sample preparation and technical challenges associated with

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Cancer Biology

Surface Engineering and Multimodal Imaging of Multistage Delivery Vectors in Metastatic Breast Cancer Authors:  Shreya Goel, Mauro Ferrari and Haifa Shen, date: 05/20/2021, view: 664, Q&A: 0

The design of effective nanoformulations that target metastatic breast cancers is challenging due to a lack of competent imaging and image analysis protocols that can capture the interactions between the injected nanoparticles and metastatic lesions. Here, we describe the integration of in vivo whole-body PET-CT with high temporal resolution, ex

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Cell Biology

FACS Enrichment of Total Interstitial Cells and Fibroblasts from Adult Mouse Ventricles Authors:  Nona Farbehi, Vaibhao Janbandhu, Robert E. Nordon and Richard P. Harvey, date: 05/20/2021, view: 925, Q&A: 0

Besides cardiomyocytes, the heart contains numerous interstitial cell types, including cardiac fibroblasts, endothelial cells, immune (myeloid and lymphoid) cells, and mural cells (pericytes and vascular smooth muscle cells), which play key roles in heart repair, regeneration, and disease. We recently published a comprehensive map of cardiac

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Developmental Biology

Imaging and Fluorescence Quantification in Caenorhabditis elegans with Flow Vermimetry and Automated Microscopy Authors:  Elissa Tjahjono, Alexey V. Revtovich and Natalia V. Kirienko, date: 05/20/2021, view: 608, Q&A: 0

Gene activation and cellular biomarkers are commonly monitored using fluorescent signals from transgenic reporters or dyes. These quantifiable markers are critical for biological research and serve as an incredibly powerful tool, even more so when combined with high-throughput screening. Caenorhabditis elegans is a particularly useful model in

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Immunology

In vitro and In vivo CD8+ T Cell Suppression Assays Authors:  Lu Xie, Guihuan Liu, Yanjun Liu and Yuming Yu, date: 05/20/2021, view: 922, Q&A: 0

CD8+CD28− T suppressor cells (Ts) have been documented to promote immune tolerance by suppressing effector T cell responses to alloantigens following transplantation. The suppressive function of T cells has been defined as the inhibitory effect of Ts on the proliferation rate of effector T cells. 3H-thymidine is a classical immunological

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Production of the Receptor-binding Domain of the Viral Spike Proteins from 2003 and 2019 SARS CoVs and the Four Common Human Coronaviruses for Serologic Assays and Inhibitor Screening Authors:  Bruno Segovia-Chumbez, Stephen D. Graham, Ramesh Jadi, Aravinda M. de Silva and Lakshmanane Premkumar, date: 05/20/2021, view: 715, Q&A: 0

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification

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A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4+ T Cells Authors:  Siwen Kang, Ruohan Wu and Ruoning Wang, date: 05/20/2021, view: 636, Q&A: 0

Functional and mechanistic studies of CD4+ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (TH17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting

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Microbiology

Parasitemia Evaluation in Mice Infected with Schistosoma mansoni Authors:  Naiara Clemente Tavares and Marina Moraes Mourão, date: 05/20/2021, view: 415, Q&A: 0

Schistosomiasis is a neglected tropical disease. Its treatment relies on the use of a single drug, praziquantel. Due to treatment limitations, an alternative for schistosomiasis chemotherapy is required; thus, a better understanding of parasite biology and host-parasite interactions is valuable to aid the identification of new anti-Schistosoma

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ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates Authors:  Thurston Herricks, Magdalena Donczew, David R. Sherman and John D. Aitchison, date: 05/20/2021, view: 406, Q&A: 0

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of

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Tracking the Subcellular Localization of Surface Proteins in Staphylococcus aureus by Immunofluorescence Microscopy Authors:  Salvatore J. Scaffidi, Mac A. Shebes and Wenqi Yu, date: 05/20/2021, view: 1128, Q&A: 0

Surface proteins of Staphylococcus aureus and other Gram-positive bacteria play essential roles in bacterial colonization and host-microbe interactions. Surface protein precursors containing a YSIRK/GXXS signal peptide are translocated across the septal membrane at mid-cell, anchored to the cell wall peptidoglycan at the cross-wall compartment,

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Molecular Biology

A New Method for Studying RNA-binding Proteins on Specific RNAs Authors:  Weiping Sun, Ziheng Zhang, Ji-Long Liu and Min Zhuang, date: 05/20/2021, view: 1146, Q&A: 0

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT.

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In vivo CD40 Silencing by siRNA Infusion in Rodents and Evaluation by Kidney Immunostaining Authors:  Miguel Hueso, Adrián Mallén, Elia Ripoll, Laura de Ramón, Nuria Bolaños, Cristian Varela, Jordi Guiteras, Javier Checa, Estanislao Navarro, Josep Maria Grinyo, Josep Maria Cruzado, Josep Maria Aran and Joan Torras, date: 05/20/2021, view: 369, Q&A: 0

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our

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Neuroscience

Operant Vapor Self-administration in Mice Authors:  Renata C. N. Marchette, Brendan J. Tunstall, Leandro F. Vendruscolo and Khaled Moussawi, date: 05/20/2021, view: 567, Q&A: 0

Models of drug addiction in rodents are instrumental in understanding the underlying neurobiology. Intravenous self-administration of drugs in mice is currently the most commonly used model; however, several challenges exist due to complications related to catheter patency. To take full advantage of the genetic tools available to study opioid

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Stem Cell

Ex vivo Tissue Culture Protocols for Studying the Developing Neocortex Authors:  Takashi Namba, Christiane Haffner and Wieland B. Huttner, date: 05/20/2021, view: 521, Q&A: 0

The size of the neocortex and its morphology are highly divergent across mammalian species. Several approaches have been utilized for the analysis of neocortical development and comparison among different species. In the present protocol (Note: This protocol requires basic knowledge of brain anatomy), we describe three ex vivo neocortical

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