1Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University
The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.
Published February 18, 2014. Keywords: Chemistry, ATP synthase, Bio-Layer Interferometry, Ligand-induced conformational change, Biomolecular Interaction Analysis, Allosteric regulation, Enzyme inhibition
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).
Published February 3, 2013. Keywords: Genetics, Molecular Biology, Bioinformatics, Cancer Biology, Pathology, Biomedical Engineering, Immunology, Intranuclear Space, Nuclear Matrix, Fluorescence in situ Hybridization, FISH, 3D DNA FISH, DNA, immunofluorescence, immuno-FISH, 3D microscopy, Nuclear organization, interphase nuclei, chromatin modifications
1Brain and Cognition, University of Amsterdam
Reading in color is a new method for training letter-color associations that are typically found only in grapheme-color synesthetes. It involves an implicit form of training that has potential for long-term associative training methods because the training is a byproduct of reading and any text can be colored.
Published February 20, 2014. Keywords: Behavior, synesthesia, training, learning, reading, vision, memory, cognition
1GE Healthcare Bio-Sciences AB
We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.
Published March 17, 2010. Keywords: Cellular Biology, Protein interaction, Surface Plasmon Resonance, Biacore X100, CFCA, Cystatin B, Papain
1Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania
The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.
Published April 19, 2013. Keywords: Molecular Biology, Genetics, Biochemistry, Microbiology, Medicine, Proteins, DNA-Binding Proteins, Transcription Factors, Chromatin Immunoprecipitation, Genes, chromatin, immunoprecipitation, ChIP, DNA, PCR, sequencing, antibody, cross-link, cell culture, assay
1Arthritis and Clinical Immunology, Oklahoma Medical Research Foundation
A protocol is described that uses Illumina's Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.
Published November 19, 2013. Keywords: Basic Protocol, genomics, SNP, Genotyping, Infinium, iScan, HiScan, Illumina
1Department of Bacteriology, University of Wisconsin-Madison
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
Published October 19, 2012. Keywords: Microbiology, Molecular Biology, Bacteriology, Developmental Biology, Colonization, Xenorhabdus, Steinernema, symbiosis, nematode, bacteria, fluorescence microscopy
JoVE Clinical and Translational Medicine
1Orvis School of Nursing, University of Nevada, Reno, 2The State University of New York at Buffalo, St. Joseph's Medical Center, 3Strong Memorial Hospital, University of Rochester Medical Center
Continuous 12-lead electrocardiographic (ECG) monitoring can identify transient myocardial ischemia, even when asymptomatic, among patients with suspected acute coronary syndrome (ACS). In this article we describe our method for initiating patient monitoring using a Holter device, downloading the ECG data for off-line analysis, and how to utilize the ECG software to identify transient ischemia.
Published December 28, 2012. Keywords: Medicine, Anatomy, Physiology, Cardiology, Myocardial Ischemia, Cardiovascular Diseases, Health Occupations, Health Care, transient myocardial ischemia, Acute Coronary Syndrome, electrocardiogram, ST-segment monitoring, Holter monitoring, research methodology
1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University
Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.
Published March 11, 2011. Keywords: Neuroscience, memory, associative learning, C. elegans, chemotaxis, spaced training, behavior
1Signalling Programme, The Babraham Institute, 2MRC Group, Cardiff School of Biosciences, Cardiff University
Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.
Published July 27, 2013. Keywords: Cellular Biology, Molecular Biology, Biochemistry, Phosphatidylinositols, Microscopy, Fluorescence, Video, Autophagy, Cell Biology, Autophagy, Omegasome, DFCP1, LC3, Live imaging, Time-lapse microscopy, cell, imaging