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
1Department of Biology, Technion - Israel Institute of Technology
Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.
Published March 14, 2014. Keywords: Biochemistry, mitochondria, mRNA localization, Yeast, S. cerevisiae, microarray, localized translation, biochemical fractionation
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
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
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
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
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
1School of Exercise and Nutrition Sciences, Deakin University
Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.
Published June 4, 2014. Keywords: Neuroscience, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
1Division of Biomaterials and Bioengineering, Department of Preventive and Restorative Dental Sciences, University of California San Francisco, 2Department of Radiology and Biomedical Imaging, University of California San Francisco, 3Xradia Inc.
In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e. geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.
Published March 7, 2014. Keywords: Bioengineering, biomechanics, bone-periodontal ligament-tooth complex, concentric loads, eccentric loads, contrast agent