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Isolation and Culture of Single Myofiber and Immunostaining of Satellite Cells from Adult C57BL/6J Mice

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In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay

Authors: G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 44, Q&A: 0

The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of ...

Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes

Authors: Jik Nijssen, Julio Aguila and Eva Hedlund, date: 07/20/2019, view: 35, Q&A: 0

Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly ...

Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line

Authors: Manasi P Jogalekar and Elba E Serrano, date: 07/20/2019, view: 29, Q&A: 0

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic ...

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In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay

Authors: G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 44, Q&A: 0

[Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...

Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes

Authors: Jik Nijssen, Julio Aguila and Eva Hedlund, date: 07/20/2019, view: 35, Q&A: 0

[Abstract] Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single ...

Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line

Authors: Manasi P Jogalekar and Elba E Serrano, date: 07/20/2019, view: 29, Q&A: 0

[Abstract] Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in in vitro culture systems have demonstrated that the use of ...

Optogenetic Inactivation of Transcription Factors in the Early Embryo of Drosophila

Authors: Stephen L. McDaniel and Melissa M. Harrison, date: 07/05/2019, view: 279, Q&A: 0

[Abstract] The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and ...

Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro

Authors: Chantal L. Márquez, Derrick Lau, James Walsh, K. M. Rifat Faysal, Michael W. Parker, Stuart G. Turville and Till Böcking, date: 07/05/2019, view: 440, Q&A: 0

[Abstract] The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence ...

Detection of Heteroplasmic Variants in the Mitochondrial Genome through Massive Parallel Sequencing

Authors: Joke Mertens, Filippo Zambelli, Dorien Daneels, Ben Caljon, Karen Sermon and Claudia Spits, date: 07/05/2019, view: 182, Q&A: 0

[Abstract] Detecting heteroplasmies in the mitochondrial DNA (mtDNA) has been a challenge for many years. In the past, Sanger sequencing was the main option to perform this analysis, however, this method could not detect low frequency heteroplasmies. Massive Parallel Sequencing (MPS) provides the opportunity to study the mtDNA in depth, but a controlled ...

In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria

Authors: Krishna K. Singh, Devendra P. Singh, Rambir Singh and Deepak K. Saini, date: 07/05/2019, view: 208, Q&A: 0

[Abstract] Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic ...

Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion

Authors: Nicola de Franceschi, Maryam Alqabandi, Winfried Weissenhorn and Patricia Bassereau, date: 07/05/2019, view: 205, Q&A: 0

[Abstract] In vitro investigation of the interaction between proteins and positively curved membranes can be performed using a classic nanotube pulling method. However, characterizing protein interaction with negatively curved membranes still represents a formidable challenge. Here, we describe our recently developed approach based on ...

Preparation and Purification of Active Recombinant Human Pancreatic Lipase in Escherichia coli

Authors: Nanami Kawaguchi, Haruko Ogawa and Kimie Date, date: 07/05/2019, view: 194, Q&A: 0

[Abstract] Human pancreatic lipase (HPL) is the main lipolytic enzyme involved in the digestion of dietary fat. An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time in an Escherichia coli (E. coli) expression system using a short Strep-tag II (ST II). The recHPL-ST II was solubilized with 8 M urea from ...

An Efficient Approach for RNA Extraction from Boar Sperm and Seminal Plasma

Authors: Vanmathy R. Kasimanickam and Ramanathan K. Kasimanickam, date: 07/05/2019, view: 210, Q&A: 0

[Abstract] Despite transcriptional silencing in mature sperm and cytoplasmic expulsion of RNA during the final sperm maturation process, thousands of RNAs have been successfully identified in ejaculated sperm. Although most of RNAs’ function is still unknown, it is suggested that sperm RNAs have a vital biological role in fertilization and post-fertilization ...

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