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Immunoblotting and Immunodetection
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the
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A Unique High‐Throughput Assay to Identify Novel Small Molecule Inhibitors of Chemotaxis and Migration
Xin‐Hua Liao, Alan R. Kimmel
Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for
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Discovering Protein‐Protein Interactions Using Nucleic Acid Programmable Protein Arrays
Yanyang Tang, Ji Qiu, Matthias Machner, Joshua LaBaer
We have developed a protocol enabling the study of protein‐protein interactions (PPIs) at the proteome level using in vitro–synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag
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RNA Whole‐Mount In Situ Hybridization Proximity Ligation Assay (rISH‐PLA), an Assay for Detecting RNA‐Protein Complexes in Intact Cells
Ioannis M. Roussis, Fiona A. Myers, Garry P. Scarlett
Techniques for studying RNA‐protein interactions have lagged behind those for DNA‐protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization—Proximity Ligation Assay (ISH‐PLA) to the study
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A Functional MicroRNA Screening Method for Organ Morphogenesis
Ivan T. Rebustini
The increasing repertoire of microRNAs expressed during organ development and their role in regulating organ morphogenesis provide a compelling need to develop methods to assess microRNA function using various in vitro and in vivo experimental models. Methods to assess microRNA function during organ
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Detecting Membrane Protein‐protein Interactions Using the Mammalian Membrane Two‐hybrid (MaMTH) Assay
Punit Saraon, Ingrid Grozavu, Sang Hyun Lim, Jamie Snider, Zhong Yao, Igor Stagljar
Protein‐protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction
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A General Non‐Radioactive ATPase Assay for Chromatin Remodeling Complexes
Benjamin Z. Stanton, Courtney Hodges, Gerald R. Crabtree, Keji Zhao
Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring
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UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis
David T. Krist, Peter K. Foote, Alexander V. Statsyuk
HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme
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Non‐Typeable Haemophilus influenzae Infection of the Junbo Mouse
Michael T. Cheeseman, Derek W. Hood
Acute otitis media, inflammation of the middle ear bulla, is the most common bacterial infection in children. For one of the principal otopathogens, non‐typeable Haemophilus influenzae (NTHi), animal models allow us to investigate host‐microbial interactions relevant to the onset and progression of
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Exploration of Inflammatory Bowel Disease in Mice: Chemically Induced Murine Models of Inflammatory Bowel Disease (IBD)
Raffaella Maria Gadaleta, Oihane Garcia‐Irigoyen, Antonio Moschetta
Inflammatory bowel disease (IBD) is a chronic multifactorial inflammatory disorder characterized by periods of activation and remission of intestinal inflammation, with potentially severe complications, that can lead to mortality. Experimental animal models of intestinal inflammation are crucial for
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A Mouse Model for Ocular Surface Staphylococcus aureus Infection
Zhiyong Zhang, Osama Abdel‐Razek, Guirong Wang
Creation of an appropriate animal model that accurately reflects the disease and host immune response to bacterial infection in humans is a major challenge in ocular‐surface infection research. For decades, mice have been the ideal small animal model for ocular‐surface infection research because of
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Cre‐loxP‐Mediated Recombination: General Principles and Experimental Considerations
Micheal A. McLellan, Nadia A. Rosenthal, Alexander R. Pinto
The cre‐ lox P–mediated recombination system (the “cre‐ lox P system”) is an integral experimental tool for mammalian genetics and cell biology. Use of the system has greatly expanded our ability to precisely interrogate gene function in the mouse, providing both spatial and temporal control of gene
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Analysis of mtDNA/nDNA Ratio in Mice
Pedro M. Quiros, Aashima Goyal, Pooja Jha, Johan Auwerx
Mitochondrial DNA (mtDNA) lacks the protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. mtDNA depletion compromises the efficient function of cells and tissues and thus impacts
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Facile Access to Bromonucleosides Using Sodium Monobromoisocyanurate (SMBI)
Jyotirmoy Maity, Smriti Srivastava, Yogesh S. Sanghvi, Ashok K. Prasad, Roger Stromberg
Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5‐Bromouridine, 5‐bromo‐2′‐deoxyuridine, 5‐bromouridine‐5′‐triphosphate, and nucleotides containing 5‐bromouridine have been tested and used for numerous biological studies. 8‐Bromopurine
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2‐Pyridinyl Thermolabile Groups as General Protectants for Hydroxyl, Phosphate, and Carboxyl Functions
Jolanta Brzezinska, Agnieszka Witkowska, Tomasz P. Kaczyński, Dominika Krygier, Tomasz Ratajczak, Marcin K. Chmielewski
Application of 2‐pyridinyl thermolabile protecting groups (2‐PyTPGs) for protection of hydroxyl, phosphate, and carboxyl functions is presented in this unit. Their characteristic feature is a unique removal process following the intramolecular cyclization mechanism and induced only by temperature
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Using Morpholinos to Control Gene Expression
Jon D. Moulton
Morpholino oligonucleotides are stable, uncharged, water‐soluble molecules used to block complementary sequences of RNA, preventing processing, read‐through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre‐mRNA, though they
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Synthesis of Geranyl‐2‐Thiouridine‐Modified RNA
Rui Wang, Phensinee Haruehanroengra, Jia Sheng
This unit describes the chemical synthesis of the S ‐geranyl‐2‐thiouridine (ges 2 U) phosphoramidite and its incorporation into RNA oligonucleotides through solid‐phase synthesis. Starting from the 2‐thiouracil nucleobase and the protected ribose, the 2‐thiouridine is synthesized and the geranyl
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Deformability Calculation for Estimation of the Relative Stability of Chemically Modified RNA Duplexes
Yoshiaki Masaki, Mitsuo Sekine, Kohji Seio
Chemical modification of RNA duplexes alters their stability. We have attempted to develop a computational approach to estimate the thermal stability of chemically modified duplexes. These studies revealed that the deformability of chemically modified RNA duplexes, calculated from molecular dynamics
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Characterization of Quadruplex DNA Structure by Circular Dichroism
Rafael del Villar‐Guerra, Robert D. Gray, Jonathan B. Chaires
Circular dichroism (CD) is a phenomenon that arises from the differential absorption of left‐ and right‐handed circularly polarized light, and may be seen with optically active molecules. CD spectroscopy provides useful spectral signatures for biological macromolecules in solution, and provides
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A Guide to Genome‐Wide Association Mapping in Plants
Liana T. Burghardt, Nevin D. Young, Peter Tiffin
Genome‐wide association studies (GWAS) have developed into a valuable approach for identifying the genetic basis of phenotypic variation. In this article, we provide an overview of the design, analysis, and interpretation of GWAS. First, we present results from simulations that explore key elements
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Jason G. Wallace, Sharon E. Mitchell
Genotyping‐by‐sequencing (GBS) refers to a suite of related methods that obtain genotype data from samples by using restriction enzyme digestion followed by high‐throughput sequencing. GBS is a refinement of restriction site–associated DNA sequencing (RADseq) methods, with a goal of being able to
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Characterization of Plant Small RNAs by Next Generation Sequencing
Sandra M. Mathioni, Atul Kakrana, Blake C. Meyers
Plant small RNAs are ∼20 to 24 nucleotide noncoding RNAs that typically have repressive regulatory roles in gene expression, functioning at the transcriptional or post‐transcriptional level. This influence on regulation of developmental and physiological processes has direct effects on phenotype.
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Whole‐Plant Manual and Image‐Based Phenotyping in Controlled Environments
Erica Agnew, Adam Bray, Eric Floro, Nate Ellis, John Gierer, César Lizárraga, Darren O'Brien, Madeline Wiechert, Todd C. Mockler, Nadia Shakoor, Christopher N. Topp
Phenotypic measurements and images of crops grown under controlled‐environment conditions can be analyzed to compare plant growth and other phenotypes from diverse varieties. Those demonstrating the most favorable phenotypic traits can then be used for crop improvement strategies. This article
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Fluorescence In Situ Hybridization for Glycine max Metaphase Chromosomes
Seth D. Findley, James A. Birchler, Gary Stacey
This article presents protocols for fluorescence in situ hybridization (FISH) in the cultivated soybean, Glycine max . The protocols represent soybean‐optimized versions developed for maize. We describe the use of two different probes types: genomic‐repeat‐based fluorescently‐tagged oligonucleotides
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Metaphase Chromosome Preparation from Soybean (Glycine max) Root Tips
Seth D. Findley, James A. Birchler, Gary Stacey
This unit presents a highly reliable protocol to produce and screen metaphase chromosome spreads from root tip cell suspensions of soybean ( Glycine max ), or other legumes. The procedures represent soybean‐optimized versions of protocols developed for maize. The use of pressurized nitrous oxide to
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Assessment of Ex Vivo Transport Function in Isolated Rodent Brain Capillaries
Gary N.Y. Chan, Ronald E. Cannon
The blood‐brain barrier plays an important role in neuroprotection; however, it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and
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The Quantitative Characterization of Functional Allosteric Effects
Terry Kenakin
Seven‐transmembrane receptors (7TMRs or GPCRs [G protein–coupled receptors]) are nature's prototypic allosteric proteins in that they mediate the interaction between ligand binding to the receptor and the receptor interacting with another cell signaling protein. A growing class of potential drugs
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Biomarkers—A General Review
Jeffrey K. Aronson, Robin E. Ferner
A biomarker is a biological observation that substitutes for and ideally predicts a clinically relevant endpoint or intermediate outcome that is more difficult to observe. The use of clinical biomarkers is easier and less expensive than direct measurement of the final clinical endpoint, and
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Logical Experimental Design and Execution in the Biomedical Sciences
Daniel J. Holder, Michael J. Marino
Lack of reproducibility has been highlighted as a significant problem in biomedical research. The present unit is devoted to describing ways to help ensure that research findings can be replicated by others, with a focus on the design and execution of laboratory experiments. Essential components for
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