1Department of Microbiology and Immunology and Department of Pathology, Queen's University, 2Ask Science Products Inc.
This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.
Published October 18, 2014. Keywords: Molecular Biology, Electroporation, Indium-Tin oxide, signal transduction, gap junctional communication, peptides, Stat3
1Department of Biochemistry, University of Leicester
The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.
Published October 16, 2014. Keywords: Biochemistry, structural biology, protein expression, recombinant protein, mammalian cell, transfection, polyethylenimine, suspension culture, affinity purification.
1Department of Biological Sciences, Rensselaer Polytechnic Institute
Mutation rates in young Saccharomyces cerevisiae cells measured through fluctuation tests are used to predict mutation frequencies for mother cells of different replicative ages. Magnetic sorting and flow cytometry are then used to measure actual mutation frequencies and age of mother cells to identify any deviations from predicted mutation frequencies.
Published October 16, 2014. Keywords: Microbiology, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
1Department of Biology, Miami University
RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.
Published October 13, 2014. Keywords: Molecular Biology, RNA interference, RNAi, gene knockdown, red flour beetle, Tribolium castaneum, injection, double-stranded RNA, functional analysis, teaching laboratories
1Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, 2Advanced Microscopy and Imaging Facility, University of Tennessee, Knoxville
Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.
Published October 13, 2014. Keywords: Plant Biology, High-pressure freezing, freeze substitution, transmission electron microscopy, ultrastructure, Nicotiana benthamiana, Arabidopsis thaliana, imaging, cryofixation, dehydration
1Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen
Though a known model, the guinea pig currently represents a niche in experimental animal sciences and limited data is available on the execution of most procedures. Here we present four different approaches to non-terminal in vivo blood sampling techniques in either conscious or anaesthetized guinea pigs.
Published October 11, 2014. Keywords: Medicine, guinea pig, animal model, blood sampling, non-terminal, saphenous, tarsal, jugular
1Department of Physics and Astronomy, VU University Amsterdam
This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.
Published October 10, 2014. Keywords: Biochemistry, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
1Division of Hematology/Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine
Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.
Published October 9, 2014. Keywords: Cellular Biology, alternative splicing, EMT, RNA, primer design, real time PCR, splice isoforms
1The RH Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, 2Earth and Life Institute, Université catholique de Louvain, 3Institut des Sciences de la Vie, Université catholique de Louvain
Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.
Published October 8, 2014. Keywords: Plant Biology, Osmotic water permeability coefficient, aquaporins, protoplasts, curve fitting, non-instantaneous osmolarity change, volume change time course
1Department of Biology, KU Leuven - University of Leuven
The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.
Published October 6, 2014. Keywords: Anatomy, Deprivation, visual system, eye, optic nerve, rodent, mouse, neuroplasticity, neuroscience