• Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011

Biochemistry

Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP Authors:  Tomoharu Kanie and Peter K. Jackson, date: 04/05/2018, view: 2362, Q&A: 0
GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct ...
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Biophysics

Xenopus laevis Oocytes Preparation for in-Cell EPR Spectroscopy Authors:  Laura John and Malte Drescher, date: 04/05/2018, view: 1304, Q&A: 0
One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into Xenopus ...
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Cancer Biology

Primary Cultures from Human GH-secreting or Clinically Non-functioning Pituitary Adenomas Authors:  Roberto Würth, Alessandra Pattarozzi, Federica Barbieri and Tullio Florio, date: 04/05/2018, view: 1246, Q&A: 0
Pituitary adenomas are among the more frequent intracranial tumors usually treated with both surgical and pharmacological–based on somatostatin and dopamine agonists–approaches. Although mostly benign tumors, the occurrence of invasive behaviors is often detected resulting in poorer prognosis. The use of primary cultures from human pituitary ...
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Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells Author:  Simona Giunta, date: 04/05/2018, view: 1666, Q&A: 0
Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers (Mitelman et al., 1997; Padilla-Nash et al., 2001). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies ...
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Cell Biology

Analysis of Mitochondrial Structure in the Body Wall Muscle of Caenorhabditis elegans Authors:  Shaarika Sarasija and Kenneth R. Norman, date: 04/05/2018, view: 1649, Q&A: 0
Mitochondrial function is altered in various pathologies, highlighting the crucial role mitochondria plays in maintaining cellular homeostasis. Mitochondrial structure undergoes constant fission and fusion in response to changing cellular environment. Due to this, analyzing mitochondrial structure could provide insight into the physiological state ...
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Developmental Biology

A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells Authors:  Stephen K Jones Jr, Eric C Spivey, James R Rybarski and Ilya J Finkelstein, date: 04/05/2018, view: 1562, Q&A: 0
Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and ...
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Immunology

Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices Authors:  Alon Oyler-Yaniv and Oleg Krichevsky, date: 04/05/2018, view: 1399, Q&A: 0
We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the ...
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Microbiology

Bacterial Competition Assay Based on Extracellular D-amino Acid Production Authors:  Laura Alvarez and Felipe Cava, date: 04/05/2018, view: 1700, Q&A: 0
Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in ...
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Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand Authors:  Shanker Shyam Sundhar Panchapakesan, Sunny C. Y. Jeng and Peter J. Unrau, date: 04/05/2018, view: 1137, Q&A: 0
A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose ...
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Neuroscience

Context-driven Salt Seeking Test (Rats) Authors:  Stephen E. Chang and Kyle S. Smith, date: 04/05/2018, view: 950, Q&A: 0
Changes in reward seeking behavior often occur through incremental learning based on the difference between what is expected and what actually happens. Behavioral flexibility of this sort requires experience with rewards as better or worse than expected. However, there are some instances in which behavior can change through non-incremental ...
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Sacral Spinal Cord Transection and Isolated Sacral Cord Preparation to Study Chronic Spinal Cord Injury in Adult Mice Authors:  Carmelo Bellardita, Maite Marcantoni, Peter Löw and Ole Kiehn, date: 04/05/2018, view: 1380, Q&A: 0
Spinal cord injury (SCI) is characterized by multiple sensory/motor impairments that arise from different underlying neural mechanisms. Linking specific sensory/motor impairments to neural mechanism is limited by a lack of direct experimental access to these neural circuits. Here, we describe an experimental model which addresses this shortcoming. ...
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Assessing Prepulse Inhibition of Startle in Mice Authors:  Christina Ioannidou, Giovanni Marsicano and Arnau Busquets-Garcia, date: 04/05/2018, view: 1869, Q&A: 0
Animal models are an important tool for studying neuropsychiatric disorders. However, a major challenge for researchers working with laboratory rodents is trying to reproduce ‘core’ symptoms of complex human disorders such as schizophrenia. Despite this challenge, however, it is still conceivable to use animal models designed to reproduce some of ...
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Measuring Spatiotemporal Dynamics of Odor Gradient for Small Animals by Gas Chromatography Authors:  Akiko Yamazoe-Umemoto, Yuishi Iwasaki and Koutarou D. Kimura, date: 04/05/2018, view: 1592, Q&A: 0
Odor is the most fundamental chemical stimulus that delivers information regarding food, mating partners, enemies, and danger in the surrounding environment. Research on odor response in animals is widespread, although studies on experimental systems in which the gradient of odor concentration is quantitatively measured has been quite limited. ...
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Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein Authors:  Shaarika Sarasija and Kenneth R. Norman, date: 04/05/2018, view: 1354, Q&A: 0
Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), ...
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Plant Science

Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq Authors:  Mauricio A. Reynoso, Germain C. Pauluzzi, Sean Cabanlit, Joel Velasco, Jérémie Bazin, Roger Deal, Siobhan Brady, Neelima Sinha, Julia Bailey-Serres and Kaisa Kajala, date: 04/05/2018, view: 2793, Q&A: 0
Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would ...
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In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts Authors:  Joo Yong Kim and Hak Soo Seo, date: 04/05/2018, view: 1634, Q&A: 0
Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude ...
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Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi Authors:  Andreas Keymer, Claudia Huber, Wolfgang Eisenreich and Caroline Gutjahr, date: 04/05/2018, view: 1450, Q&A: 0
Lipid transfer from host plants to arbuscular mycorrhiza fungi was hypothesized for several years because sequenced arbuscular mycorrhiza fungal genomes lack genes encoding cytosolic fatty acid synthase (Wewer et al., 2014; Rich et al., 2017). It was finally shown by two independent experimental approaches (Jiang et al., ...
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Fluorescein Transport Assay to Assess Bulk Flow of Molecules Through the Hypocotyl in Arabidopsis thaliana Authors:  Salva Duran-Nebreda and George W. Bassel, date: 04/05/2018, view: 1072, Q&A: 0
The bulk transport of molecules through plant tissues underpins growth and development. The stem acts as a conduit between the upper and low domains of the plant, facilitating transport of solutes and water from the roots to the shoot system, and sugar plus other elaborated metabolites towards the non-photosynthetic organs. In order to perform ...
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RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis Authors:  Sean D. Gallaher and Melissa S. Roth, date: 04/05/2018, view: 1107, Q&A: 0
Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell ...
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Evaluation of Root pH Change Through Gel Containing pH-sensitive Indicator Bromocresol Purple Authors:  Aparecida L. Silva, Keini Dressano, Paulo H. O. Ceciliato, Juan Carlos Guerrero-Abad and Daniel S. Moura, date: 04/05/2018, view: 1497, Q&A: 0
The Rapid Alkalinization Factor (RALF) is a plant hormone peptide that inhibits proton transport causing alkalinization of the extracellular media. To detect the alkalinization response elicited by RALF peptides in root cells, Arabidopsis seedlings are carefully transferred to a gel containing the pH-sensitive indicator bromocresol ...
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Stem Cell

Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 2612, Q&A: 0
For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector ...
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