1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center
This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.
Published October 23, 2014. Keywords: Biochemistry, chromatin remodeling, INO80, SNF2 family ATPase, structure-function, enzyme purification
1Harper Cancer Research Institute, 2Microbiology and Immunology, Indiana University School of Medicine, 3Department of Biological Sciences, University of Notre Dame
Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.
Published October 20, 2014. Keywords: Cellular Biology, Embryonic stem cell, Embryoid body, Hematopoietic Progenitor Cells, Retrovirus, Gene Expression, Temporal Gene Expression
1Department of Ophthalmology, Inselspital, University of Bern, 2Department of Ophthalmology, University Hospital of Basel, 3Department of Biology, University of Fribourg
Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.
Published October 20, 2014. Keywords: Cellular Biology, N-methyl-N-nitrosourea (MNU), retina, degeneration, photoreceptors, Müller cells, regeneration, zebrafish, visual function
1Department of Biological Sciences, Carnegie Mellon University
Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.
Published October 20, 2014. Keywords: Cellular Biology, Endocytosis, TIRF, total internal reflection fluorescence microscopy, clathrin, arrestin, receptors, live-cell microscopy, clathrin-mediated endocytosis
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