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Proximity‐Dependent Biotinylation for Identification of Interacting Proteins
Valerie Le Sage, Alessandro Cinti, Andrew J. Mouland
Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation.
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LOVTRAP: A Versatile Method to Control Protein Function with Light
Hui Wang, Klaus M. Hahn
We describe a detailed procedure for the use of LOVTRAP, an approach to reversibly sequester and release proteins from cellular membranes using light. In the application described here, proteins that act at the plasma membrane are held at mitochondria in the dark, and reversibly released by
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Histopathological Evaluation of Skeletal Muscle with Specific Reference to Mouse Models of Muscular Dystrophy
Rebecca L. Terry, Dominic J. Wells
The muscular dystrophies are a diverse group of degenerative diseases for which many mouse models are available. These models are frequently used to assess potential therapeutic interventions and histological evaluation of multiple muscles is an important part of this assessment. Histological
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Methodological Considerations for Optimizing and Validating Behavioral Assays
Stacey J. Sukoff Rizzo, Jill L. Silverman
Preclinical animal models are indispensable tools for translational research for which behavioral characterization and phenotyping are essential to testing hypotheses and for evaluating the potential of novel therapeutic agents to treat diseases. The methods employed for comprehensive behavioral
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Measurement of Behavioral Taste Responses in Mice: Two‐Bottle Preference, Lickometer, and Conditioned Taste‐Aversion Tests
Dany Gaillard, Jennifer M. Stratford
The natural like and dislike of foods based on taste is one of the most easily observed behaviors in animals. Animals eat palatable foods and reject aversive foods, which makes measurement of taste perception possible using various behavioral techniques. Three different methods to accurately measure
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Measurement of Intestinal and Peripheral Cholesterol Fluxes by a Dual‐Tracer Balance Method
Onne A.H.O. Ronda, Theo H. van Dijk, H.J. Verkade, Albert K. Groen
Long‐term elevated plasma cholesterol levels put individuals at risk for developing atherosclerosis. Plasma cholesterol levels are determined by the balance between cholesterol input and output fluxes. Here we describe in detail the methodology to determine the different cholesterol fluxes in mice.
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Determining Functional Aptamer‐Protein Interaction by Biolayer Interferometry
Xinhui Lou, Martin Egli, Xianbin Yang
Short single‐stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip‐and‐Read approach in which the
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Rolling Circle Amplification with Chemically Modified Nucleoside Triphosphates
Marcel Hollenstein, Masad J. Damha
Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility
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RNA Secondary Structure Prediction
David H. Mathews, Douglas H. Turner, Richard M. Watson
In this unit, protocols are provided for predicting RNA secondary structure with the user‐friendly RNAstructure desktop computer program and the RNAstructure Web server. The minimum free energy structure and a set of suboptimal structures with similar free energies are predicted. Prediction of
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Gram‐Scale Chemical Synthesis of Base‐Modified Ribonucleoside‐5′‐O‐Triphosphates
Muthian Shanmugasundaram, Annamalai Senthilvelan, Anilkumar R. Kore
This unit delineates a simple, reliable, straight‐forward, general, and efficient chemical method for the synthesis of modified nucleoside‐5′‐ O ‐triphosphates such as 5‐methylcytidine‐5′‐ O ‐triphosphate (5‐Me‐CTP), pseudouridine‐5′‐ O ‐triphosphate (pseudo‐UTP), and N 1 ‐methylpseudouridine‐5′‐ O
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Synthesis and Evaluation of Caged siRNA with Terminal Single Vitamin E Modification
Jiali Yang, Lijia Yu, Liangliang Zhang, Xingsu Long, Yuzhuo Ji, Xinjing Tang
RNA‐induced gene silencing has been widely applied as a powerful research tool in drug development due to its sequence‐specific degradation of target mRNA. Conditional regulation of gene functions with small interfering RNAs (siRNAs) is highly useful, especially when specific gene expression
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Live‐Cell Imaging of Meiotic Spindle and Chromosome Dynamics in Maize (Zea mays)
Natalie J. Nannas, R. Kelly Dawe
Live‐cell imaging is a powerful tool that allows investigators to directly observe the dynamics of cellular processes. Live imaging has proven particularly useful in studying mitotic and meiotic chromosome segregation, where the assembly of spindles and movement of chromosomes can be quantified in
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Pachytene Chromosome Preparation in Populus deltoides Marsh
Haoyang Xin, Yue Lan, Jisen Shi, Yan Ma, Mengli Xi
A technique that produces large numbers of good quality pachytene chromosome preparations has been developed for Populus . Anthers at the pachytene stage of meiosis are used as materials. There are two main modifications in our method relative to the traditional squashing method that address the
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Mass Spectrometry Imaging of Metabolites in Barley Grain Tissues
Manuela Peukert, Wai Li Lim, Udo Seiffert, Andrea Matros
Higher plants are composed of a multitude of tissues with particular functions, reflected by distinct profiles of transcripts, proteins, and metabolites. Although the rapid development of “omics” technologies has advanced plant science tremendously within recent years, analysis is frequently
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DNA Assembly for Plant Biology
Nicola J. Patron
Parallel DNA assembly methods allow multiple fragments of DNA to be compiled in a desired order in a single reaction. Several methods enable the efficient one‐step assembly of multiple DNA parts into a suitable plasmid acceptor at high efficiency. Type IIS‐mediated assembly offers the specific
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Generation of Transgenic Soybean (Glycine max) via Particle Bombardment of Embryogenic Cultures
John J. Finer
This protocol describes one method for generating transgenic soybean (Glycine max) using particle bombardment of embryogenic cultures. Embryogenic cultures consist of proliferating masses of somatic embryos and are initially obtained from the cotyledons of immature seeds. Embryogenic cultures are
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Chemical Mutagenesis of Seed and Vegetatively Propagated Plants Using EMS
Joanna Jankowicz‐Cieslak, Bradley J. Till
Chemical mutagenesis provides an inexpensive and straightforward way to generate a high density of novel nucleotide diversity in the genomes of plants and animals. Mutagenesis therefore can be used for functional genomic studies and also for plant breeding. The most commonly used chemical mutagen in
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