Volume: 8 Issue: 3 —BIO-PROTOCOL

Biochemistry

FRET-based Stoichiometry Measurements of Protein Complexes in vitro Authors: Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 7662, Q&A: 0

For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the ...

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Cell Biology

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells Authors: Adrian J. Giovannone, Elena Reales, Pallavi Bhattaram, Alberto Fraile-Ramos and Thomas Weimbs, date: 02/05/2018, view: 7616, Q&A: 0

The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on ...

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Ex vivo Analysis of Lipolysis in Human Subcutaneous Adipose Tissue Explants Authors: Pauline Decaunes, Anne Bouloumié, Mikael Ryden and Jean Galitzky, date: 02/05/2018, view: 6384, Q&A: 0

Most studies of human adipose tissue (AT) metabolism and functionality have been performed in vitro on isolated mature adipocyte or in situ using the microdialysis technique (Lafontan, 2012). However, these approaches have several limitations. The use of mature isolated adipocytes is limiting as adipocytes are not in their ...

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Measurement of Lysosomal Size and Lysosomal Marker Intensities in Adult Caenorhabditis elegans Authors: Julie M. Huynh, Hope Dang and Hanna Fares, date: 02/05/2018, view: 5957, Q&A: 0

Assays have been developed to study trafficking in various tissues of Caenorhabditis elegans. Adult C. elegans intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules ...

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Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots Author: Eugene V. Sheval, date: 02/05/2018, view: 5384, Q&A: 0

To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU), which ...

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Developmental Biology

How to Catch a Smurf? – Ageing and Beyond…
In vivo Assessment of Intestinal Permeability in Multiple Model Organisms Authors: Raquel R. Martins, Andrew W. McCracken, Mirre J. P. Simons, Catarina M. Henriques and Michael Rera, date: 02/05/2018, view: 10476, Q&A: 1

The Smurf Assay (SA) was initially developed in the model organism Drosophila melanogaster where a dramatic increase of intestinal permeability has been shown to occur during aging (Rera et al., 2011). We have since validated the protocol in multiple other model organisms (Dambroise et al., 2016) and have utilized the ...

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Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging Authors: Samuel Petshow and Marcel Wehrli, date: 02/05/2018, view: 8046, Q&A: 0

The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D ...

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Microbiology

Extraction of DNA from Murine Fecal Pellets for Downstream Phylogenetic Microbiota Analysis by Next-generation Sequencing Authors: Elien Eeckhout and Andy Wullaert, date: 02/05/2018, view: 10387, Q&A: 2

Mouse models are widely used to evaluate the potential impact of the gut microbial composition on health and disease. Standardized protocols for sampling and storing murine feces, as well as for extracting DNA from these fecal pellets are needed to limit experimental variation between different studies. Both efficient lysis of the microbiota and ...

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Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting Authors: Sara Sigurlásdóttir, Sunil D Saroj, Olaspers Sara Eriksson, Jens Eriksson and Ann-Beth Jonsson, date: 02/05/2018, view: 7173, Q&A: 0

To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted ...

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Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae Authors: Liang-Chun Wang, Qian Yu, Daniel C. Stein and Wenxia Song, date: 02/05/2018, view: 6079, Q&A: 0

Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female ...

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Molecular Biology

Determination of DNA Damage in the Retina Photoreceptors of Drosophila Authors: Damulewicz Milena and Pyza Elzbieta, date: 02/05/2018, view: 5679, Q&A: 0

The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work (Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress ...

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Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay Authors: Francesca Mattiroli, Yajie Gu and Karolin Luger, date: 02/05/2018, view: 6788, Q&A: 0

Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack ...

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Medaka-microinjection with an Upright Microscope Authors: Yu Murakami and Masato Kinoshita, date: 02/05/2018, view: 6733, Q&A: 0

We described a simple method for microinjecting DNA/RNA/Protein solutions into medaka eggs under an upright microscope. Medaka is an excellent vertebrate model for reverse genetics, because of its daily spawning, short generation time, and egg transparency. These features enable us to efficiently perform functional genomic analyses of transgenic ...

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Immunoprecipitation of Tri-methylated Capped RNA Authors: Karen E. Hayes, Jamie A. Barr, Mingyi Xie, Joan A. Steitz and Ivan Martinez, date: 02/05/2018, view: 7957, Q&A: 0

Cellular quiescence (also known as G₀ arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27^Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an ...

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Precision Tagging: A Novel Seamless Protein Tagging by Combinational Use of Type II and Type IIS Restriction Endonucleases Authors: Zhen Xu, Yan-Ning Rui, John P. Hagan and Dong H. Kim, date: 02/05/2018, view: 7383, Q&A: 0

Protein tagging is a powerful tool for performing comprehensive analyses of the biological functions of a protein of interest owing to the existence of a wide variety of tags. It becomes indispensable in some cases, such as in tracking protein dynamics in a live cell or adding a peptide epitope due to the lack of optimal antibodies. However, ...

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Neuroscience

Culture and Nucleofection of Postnatal Day 7 Cortical and Cerebellar Mouse Astroglial Cells Authors: Malek Chouchane and Marcos Romualdo Costa, date: 02/05/2018, view: 6438, Q&A: 0

Lineage reprogramming of astroglial cells isolated from different brain regions leads to the generation of different neuronal subtypes. This protocol describes the isolation and culture of neocortical and cerebellar astrocytes from postnatal mice. We also present a comprehensive description of the main steps towards successful gene delivery in ...

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Plant Science

Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis Authors: Fantao Kong and Yonghua Li-Beisson, date: 02/05/2018, view: 6935, Q&A: 0

Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been ...

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Radioactive Tracer Feeding Experiments and Product Analysis to Determine the Biosynthetic Capability of Comfrey (Symphytum officinale) Leaves for Pyrrolizidine Alkaloids Authors: Thomas Stegemann, Lars H. Kruse and Dietrich Ober, date: 02/05/2018, view: 5806, Q&A: 0

This protocol delivers a method to determine the biosynthetic capability of comfrey leaves for pyrrolizidine alkaloids independently from other organs like roots or flowers.

The protocol applies and combines radioactive tracer experiments with standard and modern techniques like thin layer chromatography (TLC), solid-phase extraction ...

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Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana Authors: Susumu Uehara, Yasuko Ito-Inaba and Takehito Inaba, date: 02/05/2018, view: 8253, Q&A: 0

In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide ...

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Systems Biology

Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) Authors: Dania M. Malik, Seth Rhoades and Aalim Weljie, date: 02/05/2018, view: 7216, Q&A: 0

Modern triple quadrupole mass spectrometers provide the ability to detect and quantify a large number of metabolites using tandem mass spectrometry (MS/MS). Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a ...

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Past Issues in 2018

Volume: 8 Issue: 3

Biochemistry

Cell Biology

Developmental Biology

Microbiology

Molecular Biology

Neuroscience

Plant Science

Systems Biology