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Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation
Dawn L. Brasaemle, Nathan E. Wolins
Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for
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Using MetaboAnalyst 3.0 for Comprehensive Metabolomics Data Analysis
Jianguo Xia, David S. Wishart
MetaboAnalyst (http://www.metaboanalyst.ca) is a comprehensive Web application for metabolomic data analysis and interpretation. MetaboAnalyst handles most of the common metabolomic data types from most kinds of metabolomics platforms (MS and NMR) for most kinds of metabolomics experiments
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DIANA‐TarBase and DIANA Suite Tools: Studying Experimentally Supported microRNA Targets
Maria D. Paraskevopoulou, Ioannis S. Vlachos, Artemis G. Hatzigeorgiou
microRNAs (miRNAs) are short non‐coding RNAs (∼22 nts) present in animals, plants, and viruses. They are considered central post‐transcriptional regulators of gene expression and are key components in a great number of physiological and pathological conditions. The accurate characterization of their
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Using the Tools and Resources of the RCSB Protein Data Bank
Luigi Di Costanzo, Sutapa Ghosh, Christine Zardecki, Stephen K. Burley
The Protein Data Bank (PDB) archive is the worldwide repository of experimentally determined three‐dimensional structures of large biological molecules found in all three kingdoms of life. Atomic‐level structures of these proteins, nucleic acids, and complex assemblies thereof are central to
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Obtaining miRNA‐Target Interaction Information from miRWalk2.0
Alisha Parveen, Norbert Gretz, Harsh Dweep
miRWalk2.0 (http://zmf.umm.uni‐heidelberg.de/mirwalk2 ) is a freely accessible, regularly updated comprehensive archive supplying the largest available collection of predicted and experimentally verified miRNA‐target interactions, with various novel and unique features to assist the scientific
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Tempest: Accelerated MS/MS Database Search Software for Heterogeneous Computing Platforms
Mark E. Adamo, Scott A. Gerber
MS/MS database search algorithms derive a set of candidate peptide sequences from in silico digest of a protein sequence database, and compute theoretical fragmentation patterns to match these candidates against observed MS/MS spectra. The original Tempest publication described these operations
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Identification of Protein Kinase Substrates by the Kinase‐Interacting Substrate Screening (KISS) Approach
Mutsuki Amano, Tomoki Nishioka, Yoshimitsu Yura, Kozo Kaibuchi
Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of
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Generation of 3D Collagen Gels with Controlled Diverse Architectures
Andrew D. Doyle
Rat tail collagen solutions have been used as polymerizable in vitro three dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. Factors such as ECM concentration, pH, ionic concentration, and temperature can alter collagen
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Nanoparticle‐Templated Molecular Recognition Platforms for Detection of Biological Analytes
Abraham G. Beyene, Gozde S. Demirer, Markita P. Landry
Molecular recognition of biological analytes with optical nanosensors provides both spatial and temporal biochemical information. A recently developed sensing platform exploits near‐infrared fluorescent single‐wall carbon nanotubes combined with electrostatically pinned heteropolymers to yield a
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Fabrication of 3‐D Reconstituted Organoid Arrays by DNA‐Programmed Assembly of Cells (DPAC)
Michael E. Todhunter, Robert J. Weber, Justin Farlow, Noel Y. Jee, Alec E. Cerchiari, Zev J. Gartner
Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three‐dimensional (3‐D) structures. The capacity to reconstitute tissues in vitro with the
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Reverse Phase Protein Arrays for Compound Profiling
Nathan Moerke, Mohammad Fallahi‐Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post‐translational modifications of interest. RPPA assays are
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Histomorphological Phenotyping of the Adult Mouse Brain
Anna Mikhaleva, Meghna Kannan, Christel Wagner, Binnaz Yalcin
This article describes a series of standard operating procedures for morphological phenotyping of the mouse brain using basic histology. Many histological studies of the mouse brain use qualitative approaches based on what the human eye can detect. Consequently, some phenotypic information may be
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Peripheral Neuropathy in Mouse Models of Diabetes
Corinne G. Jolivalt, Katie E. Frizzi, Lucie Guernsey, Alex Marquez, Joseline Ochoa, Maria Rodriguez, Nigel A. Calcutt
Peripheral neuropathy is a frequent complication of chronic diabetes that most commonly presents as a distal degenerative polyneuropathy with sensory loss. Around 20% to 30% of such patients may also experience neuropathic pain. The underlying pathogenic mechanisms are uncertain, and therapeutic
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Light and Electron Microscopy Methods for Examination of Cochlear Morphology in Mouse Models of Deafness
Andrew Parker, Lauren Chessum, Philomena Mburu, Jeremy Sanderson, Michael R. Bowl
Mice are an invaluable model organism for the study of auditory function. Even though there are differences in size and frequency response, the anatomy and physiology of the mouse and human ear are remarkably similar. In addition, the tools available for genetic manipulation in the mouse have
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Metabolic Profiling and Quantification of Neurotransmitters in Mouse Brain by Gas Chromatography‐Mass Spectrometry
Christian Jäger, Karsten Hiller, Manuel Buttini
Metabolites are key mediators of cellular functions, and have emerged as important modulators in a variety of diseases. Recent developments in translational biomedicine have highlighted the importance of not looking at just one disease marker or disease inducing molecule, but at populations thereof
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Islet Insulin Secretion Measurements in the Mouse
Alison Hugill, Kenju Shimomura, Roger D. Cox
This article describes detailed protocols for in vitro measurements of insulin function and secretion in isolated mouse islets for the analysis of glucose homeostasis. We specify a method of enzyme digestion and hand picking to isolate and release the greatest number of high quality islets from the
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Comprehensive Energy Balance Measurements in Mice
Lee Moir, Liz Bentley, Roger D. Cox
In mice with altered body composition, establishing whether it is food intake or energy expenditure, or both, that is the major determinant resulting in changed energy balance is important. In order to ascertain where the imbalance is, the acquisition of reproducible data is critical. Therefore,
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Synthesis of 4′‐Methoxy 2′‐Deoxynucleoside Phosphoramidites for Incorporation into Oligonucleotides
Magdalena Petrová, Ivan Rosenberg
This unit contains detailed synthetic protocols for the preparation of 4′‐methoxy 2′‐deoxynucleoside phosphoramidite monomers for A, G, C, T, and U. First, 3′‐silyl‐protected 2′‐deoxynucleosides (dNs) are converted in two steps to 4′,5′‐enol acetates as the key starting compounds. Next, 4′‐methoxy
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Solid‐Phase Synthesis of Oligodeoxynucleotide Analogs Containing Phosphorodithioate Linkages
Xianbin Yang
The oligodeoxynucleotide phosphorodithioate modification (PS2‐ODN) uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphordiester backbone linkage. Like a natural phosphodiester ODN backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus.
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Preparation of Intrastrand {G}O6‐Alkylene‐O6{G} Cross‐Linked Oligonucleotides
Derek K. O'Flaherty, Christopher J. Wilds
This unit describes the preparation O 6 ‐2′‐deoxyguanosine‐butylene‐ O 6 ‐2′‐deoxyguanosine dimer phosphoramidites and precursors for incorporation of site‐specific intrastrand cross‐links (IaCL) into DNA oligonucleotides. Protected 2′‐deoxyguanosine dimers are produced using the Mitsunobu reaction.
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Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry
Jingshu Guo, Robert J. Turesky
Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the
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Synthesis of Poly Linear shRNA Expression Cassettes Through Branch‐PCR
Jianbing Liu, Zhen Xi
A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch‐PCR) is described in this protocol. Double‐stranded RNA (dsRNA) is not
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Transposon Mutagenesis and Analysis of Mutants in UniformMu Maize (Zea mays)
Peng Liu, Donald R. McCarty, Karen E. Koch
A wealth of maize mutants is now available with known sites of transposon insertions in over 45% of maize genes. Materials can be obtained free of charge from the UniformMu public resource through MaizeGDB.org or directly from the Maize Genetics COOP Stock Center. Specific mutants can be sought
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Transposon Ds‐Mediated Insertional Mutagenesis in Rice (Oryza sativa)
Yuan Hu Xuan, Chul Min Kim, Byoung Il Je, Jing Miao Liu, Tian Ya Li, Gang‐Seob Lee, Tae‐Ho Kim, Chang‐Deok Han
Rice ( Oryza sativa ) is the most important consumed staple food for a large and diverse population worldwide. Since databases of genomic sequences became available, functional genomics and genetic manipulations have been widely practiced in rice research communities. Insertional mutants are the
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Telomere‐Mediated Chromosomal Truncation for Generating Engineered Minichromosomes in Maize
Nathan C. Swyers, Jon P. Cody, Morgan E. McCaw, Nathaniel D. Graham, Changzeng Zhao, Robert T. Gaeta, James A. Birchler
Minichromosomes have been generated in maize using telomere‐mediated truncation. Telomere DNA, because of its repetitive nature, can be difficult to manipulate. The protocols in this unit describe two methods for generating the telomere DNA required for the initiation of telomere‐mediated
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Preparation of Chromosomes from Zea mays
Morgan McCaw, Nathan Swyers, Nathaniel Graham, Jon Cody, Changzeng Zhao, James Birchler
High‐quality preparations of chromosomes are useful for many purposes. To prepare metaphase chromosome spreads in maize, root tips are harvested and treated with nitrous oxide to stop cell division at metaphase before being fixed in acetic acid. This process allows a high number of condensed
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Fluorescence In Situ Hybridization to Maize (Zea mays) Chromosomes
Morgan McCaw, Nathaniel Graham, Jon Cody, Nathan Swyers, Changzeng Zhao, James Birchler
Fluorescence In Situ Hybridization (FISH) is the annealing of fluorescent DNA probes to their complementary sequences on prepared chromosomes and subsequent visualization with a fluorescent microscope. In maize, FISH is useful for distinguishing each of the ten chromosomes in different accessions
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Bisulfite Sequence Analyses Using CyVerse Discovery Environment: From Mapping to DMRs
Jawon Song, Greg Zynda, Samuel Beck, Nathan M. Springer, Matthew W. Vaughn
Epigenetic modification of DNA through methylation is known to be involved in multiple biological processes such as gene suppression. However, the exact mechanism of how DNA methylations play their part is yet unclear. In mammals, CpG islands (CGI) have been studied extensively for their involvement
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Measurement of Receptor Signaling Bias
Terry Kenakin
G protein–coupled receptors (GPCRs) are often pleiotropically linked to numerous cellular signaling mechanisms in cells, and it is now known that many agonists differentially activate some signaling pathways at the expense of others. The mechanism for this effect is the stabilization of different
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A Mouse Model of Furosemide‐Induced Overactive Bladder
Michael S. Saporito, Eva Zuvich, Amy DiCamillo
Detailed in this unit is a mouse model of overactive bladder and urinary incontinence based on diuretic stress‐induced urination. The procedure involves the use of a unique, highly sensitive, and automated urine capturing method to measure urinary latency, frequency, and void volume. Although this
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In Vitro Drug Metabolism Using Liver Microsomes
Kathleen M. Knights, David M. Stresser, John O. Miners, Charles L. Crespi
Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping)
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Overview of Critical Parameters for the Design and Execution of a High‐Throughput Screen for Allosteric Ligands
Andrew Alt
Allosteric ligands modulate the activity of receptor targets by binding to sites that are distinct from the orthosteric (native agonist) binding site. Allosteric modulators have potential therapeutic advantages over orthosteric agonists and antagonists, including improved selectivity, and
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