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Past Issues in 2017

Volume: 7 Issue: 2

Biochemistry

Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model Authors:  Viviane Sant’Anna, Wanderley de Souza and Rossiane C. Vommaro, date: 01/20/2017, view: 3444, Q&A: 0
This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in ...
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Cancer Biology

In vitro Dephosphorylation Assay of c-Myc Authors:  Peng Liao, Weichao Wang and Xin Ge, date: 01/20/2017, view: 2538, Q&A: 0
This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases.
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Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime Authors:  Thomas S. Blacker, Tunde Berecz, Michael R. Duchen and Gyorgy Szabadkai, date: 01/20/2017, view: 3964, Q&A: 0
NADH and NADPH are redox cofactors, primarily involved in catabolic and anabolic metabolic processes respectively. In addition, NADPH plays an important role in cellular antioxidant defence. In live cells and tissues, the intensity of their spectrally-identical autofluorescence, termed NAD(P)H, can be used to probe the mitochondrial redox state, ...
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Developmental Biology

P-body and Stress Granule Quantification in Caenorhabditis elegans Authors:  Matthias Rieckher and Nektarios Tavernarakis, date: 01/20/2017, view: 4084, Q&A: 0
Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules is Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA ...
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Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin Authors:  Eri Shibata, Kazunori Ando and Atsushi Kawakami, date: 01/20/2017, view: 3001, Q&A: 0
Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies (Shibata et al., 2016; Yoshinari et al., 2012) is a useful ...
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Immunology

Isolation of PBMCs Using Vacutainer® Cellular Preparation Tubes (CPTTM) Authors:  Alaina Puleo, Chantia Carroll, Holden Maecker and Rohit Gupta, date: 01/20/2017, view: 8520, Q&A: 0
Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-Hypaque, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability. Cellular Preparation Tubes (CPTs) are Vacutainer blood draw tubes that contain Ficoll-Hypaque and a gel plug that ...
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Virus Binding and Internalization Assay for Adeno-associated Virus Authors:  Garrett E. Berry and Longping V. Tse, date: 01/20/2017, view: 3328, Q&A: 0
The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) ...
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Microbiology

Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry Authors:  Manish Bhuwan, Madhuri Suragani, Nasreen Z. Ehtesham and Seyed E. Hasnain, date: 01/20/2017, view: 3813, Q&A: 0
The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway ...
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Protein Expression Protocol for an Adenylate Cyclase Anchored by a Vibrio Quorum Sensing Receptor Authors:  Stephanie Beltz and Joachim E. Schultz, date: 01/20/2017, view: 2681, Q&A: 0
The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.
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Quantification of Triphenyl-2H-tetrazoliumchloride Reduction Activity in Bacterial Cells Authors:  Roberto Defez, Anna Andreozzi and Carmen Bianco, date: 01/20/2017, view: 2893, Q&A: 0
This protocol describes the use of the 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) salt to evaluate the cell redox potential of rhizobia cells. The production of brightly colored and insoluble 1,3,5-Triphenyltetrazolium formazan arising from TTC reduction is irreversible and can be easily quantified using a spectrophotometer. This protocol ...
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Neuroscience

An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo Authors:  Juan Mauricio Garré, Guang Yang, Feliksas F. Bukauskas and Michael V. L. Bennett, date: 01/20/2017, view: 3475, Q&A: 0
Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord’s highly myelinated funiculi. The ...
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Plant Science

Nitrate Assay for Plant Tissues Authors:  Lufei Zhao and Yong Wang, date: 01/20/2017, view: 4616, Q&A: 0
Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement of nitrate in plant tissues.
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In vitro Floral Induction of Cuscuta reflexa Authors:  Priyanka Das and Santilata Sahoo, date: 01/20/2017, view: 2653, Q&A: 0
Floral initiation and development in the angiosperms is the essential step on which the yield of the plant depends. Sometimes external climate or any abiotic stress hinders the floral initiation and ultimately affect the plant yield. Hence, in vitro floral induction and development can help to overcome the external climatic factor. ...
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Pseudomonas syringae Flood-inoculation Method in Arabidopsis Authors:  Yasuhiro Ishiga, Takako Ishiga, Yuki Ichinose and Kirankumar S. Mysore, date: 01/20/2017, view: 3398, Q&A: 0
Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings ...
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Bioassay of Xanthomonas albilineans Attachment on Sugarcane Leaves Authors:  Imène Mensi, Jean-Heinrich Daugrois and Philippe Rott, date: 01/20/2017, view: 3206, Q&A: 0
Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases ...
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Two-electrode Voltage-clamp Recordings in Xenopus laevis Oocytes：Reconstitution of Abscisic Acid Activation of SLAC1 Anion Channel via PYL9 ABA Receptor Authors:  Cun Wang, Jingbo Zhang and Julian I. Schroeder, date: 01/20/2017, view: 3286, Q&A: 0
Two-Electrode Voltage-Clamp (TEVC) recording in Xenopus laevis oocytes provides a powerful method to investigate the functions and regulation of ion channel proteins. This approach provides a well-known tool to characterize ion channels or transporters expressed in Xenopus laevis oocytes. The plasma membrane of the oocyte is ...
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