Volume: 3 Issue: 19 —BIO-PROTOCOL

Biochemistry

Hydroxyproline Assay Using NaBr/NaOCl Author: Derek T. A. Lamport, date: 10/05/2013, view: 8298, Q&A: 0

Hydroxyproline (Hyp) is a major constituent of a relatively few proteins that are major structural components of the extracellular matrix and primary cell wall of animals and plants respectively. Significant amounts of the cyclic amino acids proline and hydroxyproline decrease polypeptide flexibility; thus proline/hydroxyproline-rich proteins are ...

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IEF-2DE Analysis and Protein Identification Authors: Xia Wu, Steven J. Clough, Steven C. Huber and Man-Ho Oh, date: 10/05/2013, view: 13092, Q&A: 0

Isoelectric focusing followed by SDS-PAGE (IEF-2DE) separates proteins in a two-dimensional matrix of protein pI (Protein Isoelectric Point) and molecular weight (MW). The technique is particularly useful to distinguish protein isoforms (Radwan et al., 2012) and proteins that contain post-translational modifications such as ...

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Blood AST, ALT and UREA/BUN Level Analysis Authors: Yuh-Pyng Sher and Mien-Chie Hung, date: 10/05/2013, view: 30048, Q&A: 0

AST (aspartate aminotransferase; GOT, glutamate oxalacetate transaminase) and ALT (alanine aminotransferase; GPT, glutamate pyruvate transaminase) are sensitive indicators to monitor the liver function under drugs treatment or with acute viral hepatitis. The elevated AST and ALT values in the blood sample indicate liver damage or injury. The ...

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Cancer Biology

LDH-A Enzyme Assay Authors: Di Zhao, Yue Xiong, Qun-Ying Lei and Kun-Liang Guan, date: 10/05/2013, view: 11897, Q&A: 0

LDH (Lactate dehydrogenase) enzyme catalyzes the reversible conversion of pyruvate to lactate using NAD+ as a cofactor. Although the physiological significance of lactate accumulation in tumor cells, a dead-end product in cellular metabolism, is currently a topic of debate, it has long been known that many tumor cells express a high level of LDH-A ...

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Pancreatic Acinar Cell 3-Dimensional Culture Authors: Chunjing Qu and Stephen F. Konieczny, date: 10/05/2013, view: 11521, Q&A: 0

Normal pancreatic acinar cells are difficult to maintain on traditional plastic culture surfaces due to their physical properties of housing large quantities of digestive enzymes and the formation of intercellular tight junctions and gap junctions (Apte and Wilson 2005; Rukstalis et al., 2003). However, placing primary acinar cells within ...

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Cell Biology

Spindle Angle Measurements Authors: Julien Cau, Nathalie Morin and Guillaume Bompard, date: 10/05/2013, view: 9443, Q&A: 0

Spindle angles measures derive from the measures of spindle poles positions that were taken from fixed and immunostained adherent cells. To determine spindle angles (α), z-stack images of metaphasic cells immunostained with anti γ-tubulin (spindle poles) and anti β-tubulin antibodies (mitotic spindle) were acquired. A very simple ImageJ software ...

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Microbiology

Staphylococcus aureus Killing Assay of Caenorhabditis elegans Authors: Amanda C. Wollenberg, Orane Visvikis, Anna-Maria F. Alves and Javier E. Irazoqui, date: 10/05/2013, view: 10415, Q&A: 0

The Gram-positive bacterium Staphylococcus aureus is a human pathogen that displays virulence towards the nematode Caenorhabditis elegans. This property can be used to discover genes that are important for virulence in humans, because S. aureus possesses common virulence factors that are used in C. elegans and ...

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Cell Fractionation of Pseudomonas aeruginosa Authors: Esteban Paredes-Osses and Kim R. Hardie, date: 10/05/2013, view: 10051, Q&A: 1

Pseudomonas aeruginosa is a Gram negative bacterium. Separating the cell envelope compartments enables proteins to be localized to confirm where in the cell they function. Cell fractionation can also provide a first step in a protein purification strategy (Williams et al., 1998). This protocol has been designed to obtain the ...

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Virus Overlay Assay (Far-Western blotting) Authors: Kun-Tong Jia, Chang-Jun Guo, Xiao-Bo Yang and Jian-Guo He, date: 10/05/2013, view: 12118, Q&A: 0

Virus overlay assay is a method to detect protein-protein interaction in vitro. We performed the virus overlay assay to identify the receptor proteins interacting with the infectious spleen and kidney necrosis virus (ISKNV).

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Batch Culture Fermentation of Endophytic Fungi and Extraction of Their Metabolites Authors: Susheel Kumar and Nutan Kaushik, date: 10/05/2013, view: 12332, Q&A: 0

Antibiosis is one of the possible modes of action shown by endophytic fungi having antifungal activity. To test if antifungal activity in endophytic fungi is due to antibiosis, assay of the metabolites of endophytic fungi was needed. To obtain metabolites for bioassay batch culture fermentation and extraction of metabolites was done. Fungus was ...

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Bioassay of Extracts of the Endophytic Fungi Authors: Susheel Kumar and Nutan Kaushik, date: 10/05/2013, view: 7788, Q&A: 0

Many of the microbes including fungi produce metabolites possessing antifungal activity and in some cases fungal metabolites are main cause of antifungal activity resulted by fungus. To infer that antifungal activity is due to fungal metabolites, there is a need to develop a repeatable procedure to assay these metabolites. Here we are presenting ...

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Plant Science

Preparation of Arabinogalactan Glycoproteins from Plant Tissue Author: Derek T. A. Lamport, date: 10/05/2013, view: 9169, Q&A: 1

This supplements an earlier protocol (Popper, 2011) for the extraction and assay of cell surface arabinogalactan proteins (AGPs). These highly glycosylated glycoproteins (~95% carbohydrate) contain numerous glycomodules with paired glucuronic acid residues that bind Ca²⁺ in a pH dependent manner (Lamport and Varnai, 2013). Classical ...

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Measurement of Endogenous H₂O₂ and NO and Cell Viability by Confocal Laser Scanning Microscopy Authors: Mi-Mi Wu, Xian-Ge Ma and Jun-Min He, date: 10/05/2013, view: 10857, Q&A: 1

Recently, there is compelling evidence that hydrogen peroxide (H₂O₂) and nitric oxide (NO) function as signaling molecules in plants, mediating a range of responses including stomatal movement. Thus, the choice of sensitive methods for detection of endogenous H₂O₂ and NO in guard cells are very important ...

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Stomatal Bioassay in Arabidopsis Leaves Authors: Xuan Li, Xian-Ge Ma and Jun-Min He, date: 10/05/2013, view: 12299, Q&A: 1

Stomata embedded in the epidermis of terrestrial plants are important for CO₂ absorption and water transpiration, and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Furthermore, stomata can respond via accurate change of stomatal apertures to a ...

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In vitro Protein Ubiquitination Assays Authors: Qingzhen Zhao and Qi Xie, date: 10/05/2013, view: 37772, Q&A: 0

Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or ...

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MAPK Phosphorylation Assay with Leaf Disks of Arabidopsis Authors: Pascale Flury, Dominik Klauser, Thomas Boller and Sebastian Bartels, date: 10/05/2013, view: 10055, Q&A: 0

Activation of mitogen activated protein kinases (MAPKs) is involved in many abiotic and biotic stress responses including plant defense. MAPK acitvation is based on the dual phosphorylation of threonine (T) and tyrosin (Y) residues (T-x-Y motif) in the activation loop of the MAPK protein. By determination of the phosphorylation status of a ...

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Past Issues in 2013

Volume: 3 Issue: 19

Biochemistry

Cancer Biology

Cell Biology

Microbiology

Plant Science