• 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

Biochemistry

Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits Authors:  Tobias Baumann, Oliver Vosyka, Bogdan I. Florea, Hermen S. Overkleeft, Silke Meiners and Ilona E. Kammerl, date: 12/20/2016, view: 4588, Q&A: 0
Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, ...
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Cancer Biology

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes Authors:  Amaresh C Panda, Jennifer L. Martindale and Myriam Gorospe, date: 12/20/2016, view: 11994, Q&A: 0
RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al ...
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In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death Authors:  Yuting Ma and Heng Yang, date: 12/20/2016, view: 9257, Q&A: 1
Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve ...
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Cell Biology

Measurement of Mechanical Tension at cell-cell junctions using two-photon laser ablation Authors:  Xuan Liang, Magdalene Michael and Guillermo A. Gomez, date: 12/20/2016, view: 5104, Q&A: 0
The cortical actomyosin cytoskeleton is found in all non-muscle cells where a key function is to control mechanical force (Salbreux et al., 2012). When coupled to E-cadherin cell-cell adhesion, cortical actomyosin generates junctional tension that influences many aspects of tissue function, organization and morphogenesis (Lecuit and Yap, ...
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Isolation of THY1+ Undifferentiated Spermatogonia from Mouse Postnatal Testes Using Magnetic-activated Cell Sorting (MACS) Authors:  Hung-Fu Liao, Joyce Kuo, Hsien-Hen Lin and Shau-Ping Lin, date: 12/20/2016, view: 5356, Q&A: 0
In mammals, homeostasis of many tissues rely on a subpopulation of cells, referred to as stem cells, to sustain an appropriate number of undifferentiated and differentiated cells. Spermatogonial stem cells (SSCs) provide the fundamental cellular source for spermatogenesis and are responsible for the lifelong maintenance of the germline pool in ...
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Microbiology

Antibiotic Disc Assay for Synechocystis sp. PCC6803 Authors:  Otilia Cheregi and Christiane Funk, date: 12/20/2016, view: 5110, Q&A: 0
This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates ...
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Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol Authors:  Simon David Brown and Alexander Lorenz, date: 12/20/2016, view: 4629, Q&A: 0
The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described ...
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Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi Authors:  Michael Mentges and Jörg Bormann, date: 12/20/2016, view: 5023, Q&A: 0
Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense ...
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Molecular Biology

Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System Authors:  Sivasundaram Karnan, Akinobu Ota, Yuko Konishi, Md Wahiduzzaman, Shinobu Tsuzuki, Yoshitaka Hosokawa and Hiroyuki Konishi, date: 12/20/2016, view: 5124, Q&A: 0
Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide ...
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Plant Science

Detection of Reactive Oxygen Species in Oryza sativa L. (Rice) Authors:  Navdeep Kaur, Isha Sharma, Kamal Kirat and Pratap Kumar Pati, date: 12/20/2016, view: 8665, Q&A: 1
Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H2O2 is qualitatively ...
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Infection of Nicotiana benthamiana Plants with Potato Virus X (PVX) Authors:  Emmanuel Aguilar, Francisco J. del Toro, Bong-Nam Chung, Tomás Canto and Francisco Tenllado, date: 12/20/2016, view: 7030, Q&A: 0
Potato Virus X (PVX) is the type member of Potexvirus genus, a group of plant viruses with a positive-strand RNA genome (~6.4 kb). PVX is able to establish compatible infections in Nicotiana benthamiana, a commonly used host in plant virology, leading to mild symptoms, such as chlorotic mosaic and mottling. PVX has been ...
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Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1) Authors:  Joana Rocha, Valerie Chazalet and Christelle Breton, date: 12/20/2016, view: 3841, Q&A: 0
In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a ...
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Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay Authors:  Alexandre Perochon and Fiona M. Doohan, date: 12/20/2016, view: 5342, Q&A: 0
Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The ...
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Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC Authors:  László Kovács, André Vidal-Meireles, Valéria Nagy and Szilvia Z. Tóth, date: 12/20/2016, view: 4109, Q&A: 0
Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the ...
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Bacterial Growth Inhibition Assay for Xanthomonas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts Authors:  Marco Loehrer, Rhoda Delventhal, Denise Weidenbach and Ulrich Schaffrath, date: 12/20/2016, view: 4528, Q&A: 0
We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue.
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Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei) Authors:  Rhoda Delventhal, Marco Loehrer, Denise Weidenbach and Ulrich Schaffrath, date: 12/20/2016, view: 6546, Q&A: 0
To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe ...
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Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice Authors:  Erika Toda, Yukinosuke Ohnishi and Takashi Okamoto, date: 12/20/2016, view: 5032, Q&A: 0
Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation of rice gametes, electro-fusion of gametes, ...
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Protocol for Increasing Carotenoid Levels in the Roots of Citrus Plants Authors:  Matías Manzi, Marta Pitarch-Bielsa, Vicent Arbona and Aurelio Gómez-Cadenas, date: 12/20/2016, view: 4196, Q&A: 0
Carotenoids in plants play several key functions such as acting as light-harvesters, antioxidants (Lado et al., 2016) or being precursors of strigolactones, abscisic acid, volatiles and other signaling compounds (Arbona et al., 2013). Although those functions are well-known in light-exposed tissues, information in belowground ...
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Plant Tissue Trypan Blue Staining During Phytopathogen Infection Authors:  Nuria Fernández-Bautista, José Alfonso Domínguez-Núñez, M. Mar Castellano Moreno and Marta Berrocal-Lobo, date: 12/20/2016, view: 13107, Q&A: 2
In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than ever. The protocol is applied specifically to detect cell death on Arabidopsis ...
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Stem Cell

Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice Authors:  Micah T. Webster, Tyler Harvey and Chen-Ming Fan, date: 12/20/2016, view: 5166, Q&A: 0
For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during ...
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Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine Authors:  Jun Chen, Jia Li, Huaiwei Huang and Rongwen Xi, date: 12/20/2016, view: 6595, Q&A: 1
RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila ...
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