Volume: 6 Issue: 19 —BIO-PROTOCOL

Biochemistry

Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts Authors: Haydar Karakaya and Kübra Özkul, date: 10/05/2016, view: 8791, Q&A: 0

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6-phosphoglucono-δ-lactone and transfers one electron to NADP⁺ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the ...

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Measuring Rat Serum Osmolality by Freezing Point Osmometry Authors: Jerneja Stare and Charles W. Bourque, date: 10/05/2016, view: 8941, Q&A: 0

Blood serum or plasma osmolality is the measure of electrolyte to water balance in the body’s circulation, and is tightly regulated in physiological states in order to maintain normal levels of serum solute (Bourque, 2008). Osmolality is defined as the number of osmoles of solute per kg of water (mOsm/kg) (Dufour, 1993) and can be measured using ...

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PNGase Sensitivity Assay to Study the Folding Status of Proteins Authors: Satoshi Ninagawa and Kazutoshi Mori, date: 10/05/2016, view: 5022, Q&A: 0

This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is ...

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Trypsin Sensitivity Assay to Study the Folding Status of Proteins Authors: Satoshi Ninagawa and Kazutoshi Mori, date: 10/05/2016, view: 6718, Q&A: 0

This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant ...

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Cell Wall-bound p-Coumaric and Ferulic Acid Analysis Author: Nickolas Anderson, date: 10/05/2016, view: 5014, Q&A: 0

Hydroxycinnamic acids, such as p-coumaric acid and ferulic acid, are a major class of compounds derived from the phenylpropanoid pathway. These compounds are widely conserved in plants and primarily accumulate in the secondary cell wall. They serve as important structural components that contribute to the overall strength and rigidity of ...

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Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells Authors: I-Chen Peng, Alex J. Bott and Wei-Xing Zong, date: 10/05/2016, view: 8778, Q&A: 0

Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and ...

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Cancer Biology

Evaluation of Angiogenesis Inhibitors Using the HUVEC Fibrin Bead Sprouting Assay Authors: Laura Winters, Nithya Thambi, Julian Andreev and Frank Kuhnert, date: 10/05/2016, view: 9170, Q&A: 0

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is a critical process that occurs during normal development and tumor formation. Targeting tumor angiogenesis by blocking the activity of vascular endothelial growth factor (VEGF) has demonstrated some clinical benefit; nevertheless there is a great need to target additional ...

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Isolation of Primary Breast Cancer Cells from HER2 Transgenic Mice Authors: Shou Liu and Hexin Chen, date: 10/05/2016, view: 9219, Q&A: 1

HER2 is a tyrosine kinase receptor, which is overexpressed in about 30% of breast cancer patients. Its overexpression leads to mammary tumorigenesis and increased invasion and metastasis (Slamon et al., 1987). HER2 transgenic mouse (FVB/N-MMTVneu mouse) is a well-established model of mammary tumor in human (Fantozzi and Christofori, ...

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Cell Biology

Detection of Reactive Oxygen Species Using MitoSOX and CellROX in Zebrafish Authors: Alberto Rissone and Fabio Candotti, date: 10/05/2016, view: 17723, Q&A: 1

The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species (ROS) are strictly regulated by the ...

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Mouse Corneal Stroma Fibroblast Primary Cell Culture Authors: Yujin Zhang, Yen-Chiao Wang, Okada Yuka, Lingling Zhang and Chia-Yang Liu, date: 10/05/2016, view: 5727, Q&A: 0

This protocol is developed for primary cell culture of cornea stromal keratocytes isolated from neonatal mouse eyeballs. It provides an optimal condition to isolate stromal keratocytes which maintain high viability for cell culture.

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Developmental Biology

Isolation, Culture, and Staining of Single Myofibers Authors: Yann Simon Gallot, Sajedah M. Hindi, Aman K. Mann and Ashok Kumar, date: 10/05/2016, view: 9499, Q&A: 1

Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be ...

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Immunology

ASC-particle-induced Peritonitis Authors: Lucia de Almeida, Andrea Dorfleutner and Christian Stehlik, date: 10/05/2016, view: 4487, Q&A: 0

In response to pathogen infection and tissue damage, inflammasome sensors such as NLRP3 and AIM2 are activated, which triggers PYRIN domain (PYD)-mediated ASC nucleation, followed by self-perpetuating ASC polymerization, which ultimately culminates in caspase-1 activation, interleukin (IL)-1β and IL-18 processing and release and pyroptosis ...

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In vivo Analysis of Neutrophil Infiltration during LPS-induced Peritonitis Authors: Lucia de Almeida, Andrea Dorfleutner and Christian Stehlik, date: 10/05/2016, view: 6864, Q&A: 0

Bacterial lipopolysaccharide (LPS) is present in the outer membrane of Gram-negative bacteria and functions as pathogen-associated molecular pattern (PAMP) (Whitfield and Trent, 2014). LPS therefore is a potent activator of inflammatory responses leading to cytokine release and neutrophils recruitment. The lipid A moiety of LPS activates the ...

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Microbiology

Measurement of Cellular Copper in Rhodobacter capsulatus by Atomic Absorption Spectroscopy Authors: Petru-Iulian Trasnea, Dorian Marckmann, Marcel Utz and Hans-Georg Koch, date: 10/05/2016, view: 6693, Q&A: 0

Copper is an essential micronutrient and functions as a cofactor in many enzymes such as heme-Cu oxygen reductases, Cu-Zn superoxide dismutases, multi-copper oxidases and tyrosinases. However, due to its chemical reactivity, free copper is highly toxic (Rae et al., 1999) and all organisms use sophisticated machineries for controlling ...

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Molecular Biology

PCR-based Assay for Genome Integrity after Methyl Methanesulfonate Damage in Physcomitrella patens Authors: Masaki Odahara, Takayuki Inouye, Yoshiki Nishimura and Yasuhiko Sekine, date: 10/05/2016, view: 5166, Q&A: 0

In plant cells, genomic DNA exists in three organelles: the nucleus, chloroplast, and mitochondrion. Genomic DNA can be damaged by endogenous and exogenous factors, but the damaged DNA can be repaired by DNA repair systems. To quantify the extent of their repair activity of on individual genomic DNA, a PCR-based assay utilizing long amplicons is ...

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Neuroscience

Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate Authors: Fabienne C. Fiesel, Roman Hudec and Wolfdieter Springer, date: 10/05/2016, view: 5377, Q&A: 0

This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin ...

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Organotypic Spinal Cord Slice Cultures and a Method to Detect Cell Proliferation in These Slices Authors: Jillian M. Daniel, Jim Deuchars and Susan A. Deuchars, date: 10/05/2016, view: 7804, Q&A: 1

In these culture models, the normal cytoarchitecture and local neuronal circuits of the spinal cord are preserved, offering a compromise between dissociated cell cultures and complete animal studies. The addition of 5-ethynyl-2’-deoxyuridine (EdU) to the culture medium allows for the detection of proliferating cells.

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Light/Dark Transition Test to Assess Anxiety-like Behavior in Mice Authors: Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 12698, Q&A: 0

The light/dark transition test (LDT) is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in response to mild stressors, such as novel environment and light. This test is also sensitive to ...

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Sucrose Preference Test to Measure Anhedonic Behaviour in Mice Authors: Tsvetan Serchov, Dietrich van Calker and Knut Biber, date: 10/05/2016, view: 20060, Q&A: 0

The sucrose preference test (SPT) is a reward-based test, used as in indicator of anhedonia. Anhedonia, or the decreased ability to experience pleasure, represents one of the core symptoms of depression. Rodents are born with an interest in sweet foods or solutions. Reduced preference for sweet solution in SPT represents anhedonia, while this ...

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Plant Science

An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens Authors: Charlotte Gruau, Patricia Trotel-Aziz, Bas Verhagen, Sandra Villaume, Fanja Rabenoelina, Barbara Courteaux, Christophe Clément, Fabienne Baillieul and Aziz Aziz, date: 10/05/2016, view: 7819, Q&A: 0

Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One ...

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Past Issues in 2016

Volume: 6 Issue: 19

Biochemistry

Cancer Biology

Cell Biology

Developmental Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science