• Volume 10, 2020
  • Volume 9, 2019
  • Volume 8, 2018
  • Volume 7, 2017
  • Volume 6, 2016
  • Volume 5, 2015
  • Volume 4, 2014
  • Volume 3, 2013
  • Volume 2, 2012
  • Volume 1, 2011
Image Credit: BioProtoc.3651

Past Issues in 2020

Volume: 10 Issue: 12

Biochemistry

Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 1020, Q&A: 0
Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques ...
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Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity Authors:  Muthukumar Kannan, Deanna L. Davis, John Suemitsu, Christopher D. Oltorik and Binks Wattenberg, date: 06/20/2020, view: 831, Q&A: 0
Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...
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In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates Authors:  William L. Willis, Abigail Foster, Caitlin Henry, Lai Chu Wu and Wael Jarjour, date: 06/20/2020, view: 764, Q&A: 0
Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient ...
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Biophysics

Preparation of Yeast tRNA Sample for NMR Spectroscopy Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 776, Q&A: 0
Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological ...
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Cell Biology

Single Cell Volume Measurement Utilizing the Fluorescence Exclusion Method (FXm) Authors:  Nash D. Rochman, Kai Yao, Nicolas A. Perez Gonzalez, Denis Wirtz and Sean X. Sun, date: 06/20/2020, view: 957, Q&A: 0
The measurement of single cell size remains an obstacle towards a deeper understanding of cell growth control, tissue homeostasis, organogenesis, and a wide range of pathologies. Recent advances have placed a spotlight on the importance of cell volume in the regulation of fundamental cell signaling pathways including those known to orchestrate ...
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Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres Authors:  Szilárd Szikora, Tibor Novák, Tamás Gajdos, Miklós Erdélyi and József Mihály, date: 06/20/2020, view: 833, Q&A: 0
Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful ...
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Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 948, Q&A: 0
In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...
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Immunology

Assessments of HLA-I Specificities of Anti-HLA-I Monoclonal Antibodies Using Solid Phase Bead Arrays Authors:  Anita J. Zaitouna, Daniel S. Ramon and Malini Raghavan, date: 06/20/2020, view: 783, Q&A: 0
Human leukocyte antigen class I (HLA-I) molecules are a group of structurally-related cell surface proteins with a high degree of variability within the population. While only up to six variants are expressed in an individual person, the whole population contains thousands of different variants. The ability to distinguish specific variants is ...
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Microbiology

Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB) Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 997, Q&A: 0
The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined ...
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Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter Authors:  Catherine G. Triandafillou and D. Allan Drummond, date: 06/20/2020, view: 903, Q&A: 0
The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively ...
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Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9 Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 679, Q&A: 0
The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In ...
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Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 627, Q&A: 0
Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
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Neuroscience

An Alternative Maze to Assess Novel Object Recognition in Mice Authors:  José Fernando Oliveira da Cruz, Maria Gomis-Gonzalez, Rafael Maldonado, Giovanni Marsicano, Andrés Ozaita and Arnau Busquets-Garcia, date: 06/20/2020, view: 1006, Q&A: 0
The novel object recognition (NOR) task is a behavioral test commonly used to evaluate episodic-like declarative memory and it relies on the innate tendency of rodents to explore novelty. Here we present a maze used to evaluate NOR memory in mice that reduces the time of the assay while improving reliability of the measurements by increasing the ...
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Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration Authors:  Joni Vanneste, Thomas Vercruysse, Philip Van Damme, Ludo Van Den Bosch and Dirk Daelemans, date: 06/20/2020, view: 858, Q&A: 0
Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to ...
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Plant Science

Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidisArabidopsis Authors:  Fang-Yu Hwu and Martin Parniske, date: 06/20/2020, view: 799, Q&A: 0
The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification ...
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Systems Biology

Low-cost and Multiplexable Whole mRNA-Seq Library Preparation Method with Oligo-dT Magnetic Beads for Illumina Sequencing Platforms Authors:  Makoto Kashima, Ayumi Deguchi, Ayumi Tezuka and Atsushi J. Nagano, date: 06/20/2020, view: 1338, Q&A: 0
RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine ...
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