1Department of Food and Environmental Sciences, University of Helsinki, 2Institute of Biotechnology, University of Helsinki
A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.
Published April 20, 2014. Keywords: Biochemistry, Strep-tag, fusion protein, Strep-Tactin, protein complex purification, bis(sulfosuccinimidyl) suberate, BS3, protein cross-linking, protein structure stabilization, proteomics, mass spectrometry
1Fraunhofer USA Center for Molecular Biotechnology
Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.
Published April 19, 2014. Keywords: Plant Biology, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
1INSERM UMR-910, Aix-Marseille University, 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego
We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.
Published April 17, 2014. Keywords: Developmental Biology, Cell, DNA, dye injection, mouse embryo, embryo culture, ultrasound, mouse heart, stem cells
1Molecular Pharmacology and Pathology Program, Department of Pathology & Bosch Institute, University of Sydney, 2Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University
Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.
Published April 11, 2014. Keywords: Biochemistry, Vitamin C, Ascorbate, Cell swelling, Glutamate, Microplate assay, Astrocytes
1Department of Experimental Oncology, European Institute of Oncology
By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.
Published April 11, 2014. Keywords: Biochemistry, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation, histone variants, chromatome, hPTMs cross-talks
1Department of Biomedical Sciences, Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Department of Cell Biology and Anatomy, Rosalind Franklin University of Medicine and Science
Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.
Published April 8, 2014. Keywords: Developmental Biology, Zebrafish, lateral line regeneration, lateral line development, neuromasts, hair cell regeneration, disease models
1Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School
The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.
Published April 8, 2014. Keywords: Cellular Biology, skeletal muscle, muscle stem cell, satellite cell, regeneration, myoblast transplantation, muscular dystrophy, self-renewal, differentiation, myogenesis
1Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School
Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.
Published April 3, 2014. Keywords: Cellular Biology, nanopodia, TM4SF1, endothelial cell, tumor cell, F-actin, immunofluorescence staining, tetraspanin
1Department of Biological Sciences, Wayne State University
Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.
Published April 2, 2014. Keywords: Biochemistry, Chromaffin Cells, Lipid Bilayers, Microscopy, Fluorescence, Polarization, Exocytosis, membrane, TIRF, pTIRF, chromaffin, polarization, vesicle
1Institute of Laboratory Animal Science, University of Zurich, 2Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota
Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.
Published April 2, 2014. Keywords: Genetics, Oocyte microinjection, Designer nucleases, ZFN, TALEN, Genome Engineering