JoVE Biology welcomes all general biology research methodologies. Content in this section canvases all fields of cell, molecular, and organismal biology, ranging from new applications of standard techniques to novel approaches aimed at understanding the functions of life and living organisms. This diverse section includes, but is not limited to, techniques in physical biology, cellular biochemistry, genetics, physiology, systems biology and a combination of eukaryotic and prokaryotic model systems.
1Children's Hospital of Eastern Ontario Research Institute, 2Sunnybrook Research Institute, University of Toronto, 3Department of Biochemistry, University of Toronto
Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.
Published August 29, 2015. Keywords: Molecular Biology, Molecular Biology, protein phosphorylation, protein kinase, screening, signal transduction, kinomics
1Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School
De novo lipogenesis and β-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of β-fatty acid oxidation and lipogenesis.
Published August 27, 2015. Keywords: Molecular Biology, Liver, Hepatocyte, Mouse, Fatty Acid, Oxidation, Lipogenesis, Metabolism, Palmitate
1Department of Cell & Developmental Biology, SUNY Upstate Medical University
Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.
Published August 27, 2015. Keywords: Molecular Biology, cell migration, mechanosensing, durotaxis, polydimethylsiloxane, tumor invasion, extracellular matrix rigidity, mechanotransduction, U2OS
1Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, California Northstate University, 2Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University
Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.
Published August 20, 2015. Keywords: Molecular Biology, Lipophilic, insoluble, hydrophobic, drug, gut, chylomicron, VLDL, lipoprotein, enterocyte, lymph, absorption, secretion
1Molecular Neurobiology Laboratory, Psychiatry, McLean Hospital, 2Department of Biochemistry, College of Medicine, Konyang University
Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption.
Published August 17, 2015. Keywords: Biochemistry, Nurr1, Nor1, Nur77, cross reactivity, specificity, affinity purification, Nuclear receptor subfamily 4, ligand-binding domain,
1Cellular Networks Proteomics Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health
This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.
Published August 17, 2015. Keywords: Biochemistry, absolute quantification, liquid chromatography, mass spectrometry, selected reaction monitoring, stable isotope dilution series, targeted proteomics, triple quadrupole
1Centre for Cancer Research, Westmead Millennium Institute for Medical Research and University of Sydney
This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.
Published August 16, 2015. Keywords: Cellular Biology, Acute Lymphoblastic Leukemia, Cell cycle, Bromodeoxyuridine, Flow Cytometry, Chemotherapeutic agents, Cell synchronization
1Institute of Biomembranes and Bioenergetics, National Research Council
Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented. …
Published August 12, 2015. Keywords: Molecular Biology, BRCA2, siRNA, human cell lines, gene silencing, transfection, immunoblotting
1Department of Radiation and Cellular Oncology, University of Chicago, 2Department of Molecular Genetics and Cell Biology, University of Chicago
A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.
Published August 9, 2015. Keywords: Bioengineering, Budding yeast, Saccharomyces cerevisiae, cell cycle, meiosis, chromosomes, immunostaining
1Department of Genetics, Stanford University, 2Stanford Center for Genomics and Personalized Medicine, Stanford University
Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.
Published August 2, 2015. Keywords: Cellular Biology, Protein microarrays, kinase, yeast, protein-protein interactions