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Biochemistry

Analysis of the Incorporation of Carbon Atoms from Radioactive Lactate into Proteins Authors:  Tania Fiaschi and Paola Chiarugi, date: 07/05/2013, view: 3153, Q&A: 0
This method allows to analyze if the carbon atoms of lactate are embedded into proteins. Indeed, mammalian cells express the transporter of monocarboxylic acids (called MCT1) that allows the entry of lactate into the cell. To this end, cells are incubated for 24 h with the culture medium containing lactate uniformly labeled with carbon 14 and ...
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Cancer Biology

Construction of Human Monocyte Derived Macrophages Armed with Oncolytic Viruses Authors:  Munitta Muthana, Jay Richardson, Samuel Rodrigues and Claire Lewis, date: 07/05/2013, view: 4279, Q&A: 0
Macrophages are involved in many key physiological processes and complex responses such as inflammatory, immunological, infectious and neoplastic diseases. The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumours, atherosclerotic plaques, and arthritic ...
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Genomic Signature of Homologous Recombination Deficiency in Breast and Ovarian Cancers Authors:  Tatiana Popova, Elodie Manié and Marc-Henri Stern , date: 07/05/2013, view: 6720, Q&A: 0
Homologous recombination deficiency, mainly resulted from BRCA1 or BRCA2 inactivation (so called BRCAness), is found in breast and ovarian cancers. Detection of actual inactivation of BRCA1/2 in a tumor is important for patients’ treatment and follow-up as it may help predicting response to DNA damaging agents and give ...
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Cell Biology

cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer) Authors:  Cassandra Koole, Denise Wootten and Patrick Sexton, date: 07/05/2013, view: 6252, Q&A: 0
Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ...
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pERK Detection Assays Using the Surefire AlphaScreen® Kit (TGR Biosciences and PerkinElmer) Authors:  Cassandra Koole, Denise Wootten and Patrick Sexton, date: 07/05/2013, view: 4813, Q&A: 0
Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are serine/threonine protein kinases that are phosphorylated on Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2). Phosphorylation of ERK1/2 (pERK1/2) arises from multiple stimuli, resulting in physiological responses that include cell growth, proliferation and differentiation. This protocol has ...
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Detection of Released CO2 by Radioactive Lactate Authors:  Tania Fiaschi and Paola Chiarugi, date: 07/05/2013, view: 3550, Q&A: 0
This method allows to evaluate the degradation of lactate during cellular respiration. During this metabolic process, carbon atoms of lactate can be transformed in carbon dioxide. For this purpose, the radioactive lactate is added to the cells and the amount of radioactive carbon dioxide liberated is monitored. The radioactive carbon dioxide ...
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MACS Isolation and Culture of Mouse Liver Mesothelial Cells Authors:  Yuchang Li, Ingrid Lua and Kinji Asahina, date: 07/05/2013, view: 4525, Q&A: 0
Mesothelial cells (MCs) form a single squamous epithelial cell layer and cover the surfaces of the internal organs, as well as the walls of cavities. The isolation of MCs is of great importance to study their function and characteristics for the understanding of physiology and pathophysiology of the liver. Glycoprotein M6a (GPM6A) was originally ...
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Transmission Electron Microscopy (TEM) Protocol: Observation Details within Cells Authors:  Wei-Haw Peng, Kuo-Shyan Lu, Shu-Mei Lai, Horng-Tzer Shy and Hsiu-Ni Kung, date: 07/05/2013, view: 4732, Q&A: 0
Transmission electron microscopy is a technique for observing the fine details of organelles in cells or tissues. This protocol is to be used to exam the membrane structure in cells with or without virus infection. Modifications should be made if users want to get images from tissues.
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Isolation, Culture and Differentiation of Primary Acinar Epithelial Explants from Adult Murine Pancreas Author:  Clara Lubeseder-Martellato, date: 07/05/2013, view: 5498, Q&A: 0
The adult pancreas possesses an intrinsic developmental plasticity whereby acinar cells can convert into ductal structures under some pathological conditions. Acinar tissue can be isolated from murine pancreas and kept in three-dimensional collagen culture. Acinar to ductal metaplasia can be induced in primary acinar epithelial explants by ...
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Microbiology

Bacterial Conjugation in Rhodobacter capsulatus Authors:  Molly M. Leung and John Thomas Beatty, date: 07/05/2013, view: 5893, Q&A: 0
Bacterial conjugation of plasmids is the common method of introducing foreign DNA into Rhodobacter capsulatus because transformational systems have not been shown as efficient methods of introducing DNA to R. capsulatus. For R. capsulatus bacterial conjugation using an Escherichia coli donor can be used to ...
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Preparation of Pneumococcal Proteins for Western Blot Analysis Authors:  Maria João Frias, José Melo-Cristino and Mário Ramirez, date: 07/05/2013, view: 4074, Q&A: 0
This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to ...
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RNA-Affinity Chromatography Authors:  Lucia Morales , Pedro A. Mateos-Gomez, Luis Enjuanes and Isabel Sola, date: 07/05/2013, view: 4858, Q&A: 0
RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The ...
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Plant Science

Fluorescent Dye Based Measurement of Vacuolar pH and K+ Authors:  Elias Bassil, Melanie Krebs, Stephen Halperin*, Karin Schumacher and Eduardo Blumwald, date: 07/05/2013, view: 6054, Q&A: 0
Availability of ion specific fluorescent dyes has enabled the possibility to perform in vivo ion specific measurements using live cell imaging in many cellular compartments (Krebs et al., 2010; Bassil et al., 2011; Halperin and Lynch, 2003; Swanson et al., 2011; O'Connor and Silver, 2007). The importance of ion ...
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Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay Authors:  Wen-Yu Kuo, Chien-Hsun Huang, Chun Shih and Tsung-Luo Jinn, date: 07/05/2013, view: 10750, Q&A: 2
Superoxide dismutase (SOD) acts as a primary defence against reactive oxygen species (ROS) by converting O2- to O2 and H2O2. Members of this enzyme family include CuZnSOD, MnSOD and FeSOD. Most eukaryotes harbor CuZnSOD and MnSOD, and FeSOD is found in plants and prokaryotes. This protocol is to ...
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