Most read Protocols
Computational Analysis and Phylogenetic Clustering of SARS-CoV-2 Genomes
Authors:  Bani Jolly and Vinod Scaria, date: 04/20/2021, view: 1469, Q&A: 0

COVID-19, the disease caused by the novel SARS-CoV-2 coronavirus, originated as an isolated outbreak in the Hubei province of China but soon created a global pandemic and is now a major threat to healthcare systems worldwide. Following the rapid human-to-human transmission of the infection, institutes around the world have made efforts to generate

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Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase
Authors:  Abhishek Chhetri, Anna Loksztejn and Kenichi Yokoyama, date: 04/20/2021, view: 1162, Q&A: 0

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely

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Screening for Lysogen Activity in Therapeutically Relevant Bacteriophages
Authors:  Fernando L. Gordillo Altamirano and Jeremy J. Barr, date: 04/20/2021, view: 1498, Q&A: 1

Lysogenic phages can integrate into their bacterial host’s genome, potentially transferring any genetic information they possess including virulence or resistance genes, and are therefore routinely excluded from therapeutic applications. Lysogenic behavior is typically seen in phages that create turbid plaques or possess subpar bactericidal

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A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 1091, Q&A: 0

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

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Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension

Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

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Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
Authors:  Linlin Yang, Alison M. Rojas and Celia E. Shiau, date: 03/20/2021, view: 1259, Q&A: 0

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development.

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Multilayered Fabrication Assembly Technique to Engineer a Corneal Stromal Equivalent
Authors:  Julia Fernández-Pérez, Peter W. Madden and Mark Ahearne, date: 03/20/2021, view: 1032, Q&A: 0

Tissue engineering has emerged as a strategy to combat the donor shortage of human corneas for transplantation. Synthetic corneal substitutes are currently unable to support the normal phenotype of human cells and so decellularized animal corneas have been deployed to more closely provide the topographical and biochemical cues to promote cell

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Intestinal Co-culture System to Study TGR5 Agonism and Gut Restriction
Authors:  Snehal N. Chaudhari and A. Sloan Devlin, date: 03/20/2021, view: 1254, Q&A: 0

The activation of the Takeda G-protein receptor 5 (TGR5, also known as the G protein-coupled bile acid receptor 1, GPBAR1) in enteroendocrine L-cells results in secretion of the anti-diabetic hormone Glucagon-Like Peptide 1 (GLP-1) into systemic circulation. Consequently, recent research has focused on identification and development of TGR5

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Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells
Authors:  Mehrshad Pakdel, Natalia Pacheco-Fernandez and Julia von Blume, date: 03/20/2021, view: 1089, Q&A: 0

More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway in order to mature and be properly sorted to their final destinations. Among several methodologies that describe live-cell monitoring of vesicles, the Retention Using Selective Hooks (RUSH) system is a powerful one that allows to visualize

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Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric

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Murine Monocyte and Macrophage Culture
Authors:  Simone M. Haag and Aditya Murthy, date: 03/20/2021, view: 2369, Q&A: 0

Myeloid progenitors in the bone marrow generate monocytes, macrophages, granulocytes and most dendritic cells. Even though these innate immune cells are part of the same lineage, each cell type plays a specific and critical role in tissue development, host defense and the generation of adaptive immunity. Protocols have been developed in the past

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Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids
Authors:  Daniel C. Volke, Nicolas T. Wirth and Pablo I. Nikel, date: 02/20/2021, view: 2250, Q&A: 0

Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ

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Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies
Authors:  Wenjie Zhao, Xudong Wang and Libin Ye, date: 02/20/2021, view: 1926, Q&A: 0

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains,

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Evaluation of the Sequence Variability within the PCR Primer/Probe Target Regions of the SARS-CoV-2 Genome
Authors:  Kashif Aziz Khan and Peter Cheung, date: 12/20/2020, view: 1623, Q&A: 0
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it ...
Native Co-immunoprecipitation Assay to Identify Interacting Partners of Chromatin-associated Proteins in Mammalian Cells
Authors:  Afzal Husain, Nasim A. Begum, Maki Kobayashi and Tasuku Honjo, date: 12/05/2020, view: 1959, Q&A: 0

Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to

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Agrobacterium-mediated Transformation of Sweet Basil (Ocimum basilicum)
Authors:  Natasha Navet and Miaoying Tian, date: 11/20/2020, view: 1894, Q&A: 1

Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we

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Colorimetric RT-LAMP Methods to Detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic ...
Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
Authors:  Yu Jin and Sebastian Marquardt, date: 10/20/2020, view: 2238, Q&A: 0
CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an ...
Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides
Authors:  Kalina T. Haas, Methieu Rivière, Raymond Wightman and Alexis Peaucelle, date: 10/05/2020, view: 2294, Q&A: 0
The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these ...
A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
Authors:  Natarajan V. Bhanu, Simone Sidoli and Benjamin A Garcia, date: 09/20/2020, view: 2367, Q&A: 0
Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have ...
Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells
Authors:  Kevin Bode, Corinna Link, Peter H. Krammer and Heiko Weyd, date: 09/05/2020, view: 2738, Q&A: 0
Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation ...
In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin
Authors:  Michael R. Mazzucco, Timothy Vartanian and Jennifer R. Linden, date: 08/05/2020, view: 2345, Q&A: 0
In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological ...
Measuring Intracellular Vesicle Density and Dispersion Using Fluorescence Microscopy and ImageJ/FIJI
Cell signalling, cell secretion, and plasma membrane repair are processes that critically rely on intracellular vesicles, important components of the endocytic and secretory pathways. More specifically, the strategic distribution of intracellular vesicles is important for diverse cellular processes. The method presented here is a simple, ...
Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations
Authors:  Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 4448, Q&A: 0
Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...
Mouse Adipose Tissue Protein Extraction
Authors:  Yu A. An and Philipp E. Scherer, date: 06/05/2020, view: 4102, Q&A: 0
As obesity becomes a global epidemic, the metabolism research field is increasingly focusing on studying the physiological and pathological roles of adipose tissues (AT). However, extracting proteins from AT is challenging due to abundant fat content of intracellular lipid droplets. Several commercial kits for extraction of AT proteins are ...
Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
Authors:  Regina Wirth, Peng Gao, G. Ulrich Nienhaus, Murat Sunbul and Andres Jäschke, date: 05/05/2020, view: 4099, Q&A: 0
Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be ...
Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 4465, Q&A: 0
Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...
Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide
Authors:  Anthony Yammine, Jinlong Gao and Ann H. Kwan, date: 06/05/2019, view: 12410, Q&A: 0
Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. ...
Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method
Authors:  Shraddha Gang, Sheetal Sharma, Meenu Saraf, Martin Buck and Jörg Schumacher, date: 05/05/2019, view: 15126, Q&A: 1
Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a ...
Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells
Authors:  Gal Haimovich and Jeffrey E. Gerst, date: 11/05/2018, view: 23282, Q&A: 1
Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects ...
mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells
Authors:  Madara Ratnadiwakara and Minna-Liisa Änkö, date: 11/05/2018, view: 20699, Q&A: 2
Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs ...
Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
Authors:  Birte Plitzko and Sandra Loesgen, date: 05/20/2018, view: 56385, Q&A: 0
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual ...
Microbial Mutagenicity Assay: Ames Test
Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 25995, Q&A: 2
The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...
Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice
Authors:  Patrícia A. Correia, Sara Matias and Zachary F. Mainen, date: 09/20/2017, view: 13501, Q&A: 0
Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the ...
Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
Authors:  Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 30119, Q&A: 0
Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...
Extraction of Soluble and Insoluble Protein Fractions from Mouse Brains and Spinal Cords
Author:  Oliver Wirths, date: 08/05/2017, view: 21303, Q&A: 1
The current protocol details the preparation of soluble and insoluble protein lysates from mouse brain or spinal cord samples. In detail, tissue homogenization and sequential protein extraction are described. This procedure yields soluble and insoluble protein extracts that can be further processed in down-stream applications like ELISA or Western ...
Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader
Authors:  Magdiel Martínez, Namyr A. Martínez and Walter I. Silva, date: 07/20/2017, view: 25809, Q&A: 1
Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured ...
BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry
Authors:  Bo Qiu and M. Celeste Simon, date: 09/05/2016, view: 45706, Q&A: 0
Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis ...
Minimal Inhibitory Concentration (MIC) Assay for Acinetobacter baumannii
Authors:  Ming-Feng Lin, Yun-You Lin and Chung-Yu Lan, date: 12/05/2014, view: 43328, Q&A: 3
Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after overnight incubation. MIC determination is used as not only a diagnostic tool in treating bacterial infections for clinicians but also a research method in evaluating the efficacy of an ...
Dimethylmethylene Blue Assay (DMMB)
Authors:  Vivien Jane Coulson- Thomas and Tarsis Ferreira Gesteira, date: 09/20/2014, view: 49997, Q&A: 8
Glycosaminoglycans (GAGs) are long unbranched polysaccharides consisting of repeating disaccharide units composed of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (glucuronic or iduronic acid). Depending on the disaccharide unit the GAGs can be organized into five groups: chondroitin sulfate, dermatan sulfate, heparan sulfate, ...



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