Electrophoresis, Gel, Two-Dimensional:
Electrophoresis in which a second perpendicular electrophoretic transport is performed on the separate components resulting from the first electrophoresis. This technique is usually performed on polyacrylamide gels.
JoVE Immunology and Infection
1Department of Microbiology, Immunology and Pathology, Colorado State University
Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.
Published July 15, 2011. Keywords: Immunology, Mycobacterium leprae, leprosy, biopsy, STR, VNTR, PCR, fragment length analysis
1Department of Biology, University of Florida, 2UF Genetics Institute, University of Florida, 3Plant Molecular & Cellular Biology Program, University of Florida
Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.
Published February 12, 2012. Keywords: Basic Protocols, Molecular Biology, minigel, cassettes, protein, gel, electrophoresis
1Epigenetics Group, Cancer Research Program, Garvan Institute of Medical Research, 2St Vincent's Clinical School, University of NSW
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
Published October 21, 2011. Keywords: Genetics, epigenetics, DNA methylation, Bisulphite, 5-methylcytosine (5-MeC), PCR
1Institute for Biophysical Dynamics, University of Chicago, 2Physics Department - James Franck Institute, University of Chicago, 3Interdisciplinary Scientist Training Program, University of Chicago
In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.
Published December 14, 2010. Keywords: Bioengineering, Traction force microscopy, cellular adhesion, polyacrylamide gel, stiffness, elastic modulus
1Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen
Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.
Published November 16, 2013. Keywords: Basic Protocols, FtsZ, protein polymerization, cell division, GTPase, sedimentation assay, light scattering
1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine
The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.
Published November 23, 2009. Keywords: Developmental Biology, zebrafish, regeneration, barbel, surgery, vasculature, circulation, imaging, agar, embedding, microscopy
1Pall Life Sciences
This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.
Published January 5, 2011. Keywords: Molecular Biology, Plasmid purification, High-throughput, miniprep, filter plates
1Protein Expression and Purification Core Facility, EMBL Heidelberg
A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.
Published August 14, 2009. Keywords: Basic Protocols, SDS-PAGE, Coomassie staining, Protein detection, Protein staining
1Department of Nephrology, Children's Hospital of Pittsburgh of UPMC, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.
Published December 13, 2013. Keywords: Basic Protocol, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco
Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.
Published September 27, 2013. Keywords: Bioengineering, Nervous System Diseases, Surgical Procedures, Operative, Investigative Techniques, Nonmetallic Materials, Engineering (General), neural interfaces, polymer neural probes, surgical insertion, polyethylene glycol, microelectrode arrays, chronic implantation